1: FEBS J. 2008 May;275(10):2632-43. Epub 2008 Apr 17.

Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic
archaeon in a dimeric form.

Koga Y, Katsumi R, You DJ, Matsumura H, Takano K, Kanaya S.

Department of Material and Life Science, Graduate School of Engineering, Osaka
University, Japan.

The crystal structure of glycerol kinase from the hyperthermophilic archaeon
Thermococcus kodakaraensis (Tk-GK) in a dimeric form was determined at a
resolution of 2.4 A. This is the first crystal structure of a hyperthermophilic
glycerol kinase. The overall structure of the Tk-GK dimer is very similar to that
of the Escherichia coli glycerol kinase (Ec-GK) dimer. However, two dimers of
Ec-GK can associate into a tetramer with a twofold axis, whereas those of Tk-GK
cannot. This may be the reason why Tk-GK is not inhibited by fructose
1,6-bisphosphate, because the fructose 1,6-bisphosphate binding site is produced 
only when a tetrameric structure is formed. Differential scanning calorimetry
analyses indicate that Tk-GK is a highly thermostable protein with a melting
temperature (T(m)) of 105.4 degrees C for the major transition. This value is
higher than that of Ec-GK by 34.1 degrees C. Comparison of the crystal structures
of Tk-GK and Ec-GK indicate that there is a marked difference in the number of
ion pairs in the alpha16 helix. Four ion pairs, termed IP1-IP4, are formed in
this helix in the Tk-GK structure. To examine whether these ion pairs contribute 
to the stabilization of Tk-GK, four Tk-GK and four Ec-GK derivatives with
reciprocal mutations at the IP1-IP4 sites were constructed. The determination of 
their stabilities indicates that the removal of each ion pair does not affect the
stability of Tk-GK significantly, whereas the mutations designed to introduce one
of these ion pairs stabilize or destabilize Ec-GK considerably. These results
suggest that the ion pairs in the alpha16 helix contribute to the stabilization
of Tk-GK in a cooperative manner.

PMID: 18422647 [PubMed - in process]

2: Int J Biol Sci. 2008 Mar 3;4(2):71-80.

Shedding light on the role of Vitreoscilla hemoglobin on cellular catabolic
regulation by proteomic analysis.

Isarankura-Na-Ayudhya C, Panpumthong P, Tangkosakul T, Boonpangrak S,
Prachayasittikul V.

Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol
University, Bangkok 10700, Thailand.

Heterologous expression of Vitreoscilla hemoglobin (VHb) has been reported to
improve cell growth, protein synthesis, metabolite productivity and nitric oxide 
detoxification. Although it has been proposed that such phenomenon is attributed 
to the enhancement of respiration and energy metabolism by facilitating oxygen
delivery, the mechanism of VHb action remains to be elucidated. In the present
study, changes of protein expression profile in Escherichia coli as a consequence
of VHb production was investigated by two-dimensional gel electrophoresis (2-DE) 
in conjunction with peptide mass fingerprinting. Total protein extracts derived
from cells expressing native green fluorescent protein (GFPuv) and chimeric
VHbGFPuv grown in Luria-Bertani broth were prepared by sonic disintegration. One 
hundred microgram of proteins was individually electrophoresed in IEF-agarose rod
gels followed by gradient SDS-PAGE gels. Protein spots were excised from the
gels, digested to peptide fragments by trypsin, and analyzed using
matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass
spectrometry. Results revealed that expression of VHbGFPuv caused an entire
disappearance of tryptophanase as well as down-regulated proteins involved in
various metabolic pathways, e.g. glycerol kinase, isocitrate dehydrogenase,
aldehyde dehydrogenase, and D-glucose-D-galactose binding protein. Phenotypic
assay of cellular indole production confirmed the differentially expressed
tryptophanase enzymes in which cells expressing chimeric VHbGFP demonstrated a
complete indole-negative reaction. Supplementation of delta-aminolevulinic acid
(ALA) to the culture medium enhanced expression of glyceraldehyde-3-phosphate
dehydrogenase and glycerol kinase. Our findings herein shed light on the
functional roles of VHb on cellular carbon and nitrogen consumptions as well as
regulation of other metabolic pathway intermediates, possibly by autoregulation
of the catabolite repressor regulons.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 18345284 [PubMed - indexed for MEDLINE]

3: Biochemistry. 2007 Oct 30;46(43):12355-65. Epub 2007 Oct 9.

IIAGlc inhibition of glycerol kinase: a communications network tunes protein
motions at the allosteric site.

Yu P, Lasagna M, Pawlyk AC, Reinhart GD, Pettigrew DW.

Department of Biochemistry and Biophysics and Texas Agricultural Experiment
Station, Texas A&M University, College Station, Texas 77843-2128, USA.

Steady-state and time-resolved fluorescence anisotropy methods applied to an
extrinsic fluorophore that is conjugated to non-native cysteine residues
demonstrate that amino acids in an allosteric communication network within a
protein subunit tune protein backbone motions at a distal site to enable
allosteric binding and inhibition. The unphosphorylated form of the
phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli
glycerol kinase, binding more than 25 A from the kinase active site. Crystal
structures that showed a ligand-dependent conformational change and large
temperature factors for the IIAGlc-binding site on E. coli glycerol kinase
suggest that motions of the allosteric site have an important role in the
inhibition. Three E. coli glycerol kinase amino acids that are located at least
15 A from the active site and the allosteric site were shown previously to be
necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol
kinase from Haemophilus influenzae. These three amino acids are termed the
coupling locus. The apparent allosteric site motions and the requirement for the 
distant coupling locus to transplant allosteric inhibition suggest that the
coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this
possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. 
influenzae glycerol kinase were constructed with a non-native cysteine residue
replacing one of the native residues in the IIAGlc-binding site. The extrinsic
fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated
specifically to the non-native cysteine residue. Steady-state and time-resolved
fluorescence anisotropy measurements show that the motions of the fluorophore
reflect backbone motions of the IIAGlc-binding site and these motions are
modulated by the amino acids at the coupling locus.

Publication Types: 
    Research Support, N.I.H., Extramural
    Research Support, Non-U.S. Gov't

PMID: 17924663 [PubMed - indexed for MEDLINE]

4: Biochemistry. 2007 May 15;46(19):5722-31. Epub 2007 Apr 19.

Crystal structure of a hyperactive Escherichia coli glycerol kinase mutant Gly230
--> Asp obtained using microfluidic crystallization devices.

Anderson MJ, DeLabarre B, Raghunathan A, Palsson BO, Brunger AT, Quake SR.

Department of Biochemistry and Molecular Biophysics, California Institute of
Technology, MS 128-95, Pasadena, California 91125, USA.

The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 -->
Asp (GKG230D) was determined to 2.0 A resolution using a microfluidics based
crystallization platform. The crystallization strategy involved a suite of
microfluidic devices that characterized the solubility trends of GKG230D,
performed nanoliter volume free interface diffusion crystallization experiments, 
and produced diffraction-quality crystals for in situ data collection. GKG230D
displays increased enzymatic activity and decreased allosteric regulation by the 
glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to
wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric
regulation is a result of the altered FBP binding loop conformations in GKG230D
that interfere with the wild-type FBP binding site. The altered FBP binding loop 
conformations in GKG230D are supported through a series of intramolecular loop
interactions. The appearance of Asp230 in the FBP binding loops also repositions 
the wild-type FBP binding residues away from the FBP binding site. Light
scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer
formation in the presence of FBP, whereas GKWT dimers are converted into
putatively inactive tetramers in the presence of FBP. GKG230D also provides the
first structural evidence for multiple GK monomer conformations in the presence
of glycerol and in the absence of a nucleotide substrate and verifies that
glycerol binding is not responsible for locking GK into the closed conformation
necessary for GK activity.

Publication Types: 
    Research Support, N.I.H., Extramural
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.

PMID: 17441732 [PubMed - indexed for MEDLINE]

5: J Mol Biol. 2007 Jan 19;365(3):783-98. Epub 2006 Oct 25.

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia
coli.

Di Luccio E, Petschacher B, Voegtli J, Chou HT, Stahlberg H, Nidetzky B, Wilson
DK.

Section of Molecular and Cellular Biology, University of California, Davis, CA
95616, USA.

The primary metabolic route for D-xylose, the second most abundant sugar in
nature, is via the pentose phosphate pathway after a two-step or three-step
conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17)
phosphorylates D-xylulose, the last step in this conversion. The apo and
D-xylulose-bound crystal structures of Escherichia coli XK have been determined
and show a dimer composed of two domains separated by an open cleft. XK
dimerization was observed directly by a cryo-EM reconstruction at 36 A
resolution. Kinetic studies reveal that XK has a weak substrate-independent
MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with
low catalytic efficiency. Binding of pentulose and MgATP to form the reactive
ternary complex is strongly synergistic. Although the steady-state kinetic
mechanism of XK is formally random, a path is preferred in which D-xylulose binds
before MgATP. Modelling of MgATP binding to XK and the accompanying
conformational change suggests that sugar binding is accompanied by a dramatic
hinge-bending movement that enhances interactions with MgATP, explaining the
observed synergism. A catalytic mechanism is proposed and supported by relevant
site-directed mutants.

Publication Types: 
    Research Support, N.I.H., Extramural
    Research Support, Non-U.S. Gov't

PMID: 17123542 [PubMed - indexed for MEDLINE]

6: J Mol Biol. 2006 Jun 9;359(3):787-97. Epub 2006 Apr 25.

Structure and reaction mechanism of L-rhamnulose kinase from Escherichia coli.

Grueninger D, Schulz GE.

Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität,
Albertstr. 21, 79104 Freiburg im Breisgau, Germany.

Bacterial L-rhamnulose kinase participates in the degradation of L-rhamnose,
which is ubiquitous and particularly abundant in some plants. The enzyme
catalyzes the transfer of the gamma-phosphate group from ATP to the 1-hydroxyl
group of L-rhamnulose. We determined the crystal structures of the substrate-free
kinase and of a complex between the enzyme, ADP and L-fructose, which besides
rhamnulose is also processed. According to its chainfold, the kinase belongs to
the hexokinase-hsp70-actin superfamily. The closest structurally known homologue 
is glycerol kinase. The reported structures reveal a large conformational change 
on substrate binding as well as the key residues involved in catalysis. The
substrates ADP and beta-L-fructose are in an ideal position to define a direct
in-line phosphoryl transfer through a bipyramidal pentavalent intermediate. The
enzyme contains one disulfide bridge at a position where two homologous glycerol 
kinases are regulated by phosphorylation and effector binding, respectively, and 
it has two more pairs of cysteine residues near the surface that are poised for
bridging. However, identical catalytic rates were observed for the enzyme in
reducing and oxidizing environments, suggesting that regulation by disulfide
formation is unlikely.

PMID: 16674975 [PubMed - indexed for MEDLINE]

7: J Biol Chem. 2006 May 5;281(18):12833-40. Epub 2006 Mar 9.

Topography of the surface of the signal-transducing protein EIIA(Glc) that
interacts with the MalK subunits of the maltose ATP-binding cassette transporter 
(MalFGK2) of Salmonella typhimurium.

Blüschke B, Volkmer-Engert R, Schneider E.

Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin,
Chausseestr. 117, D-10115 Berlin, Germany.

The signal-transducing protein EIIA(Glc), a component of the
phosphoenolpyruvate-glucose phosphotransferase system, plays a key role in carbon
regulation in enteric bacteria, such as Escherichia coli and Salmonella
typhimurium. The phosphorylation state of EIIA(Glc) governs transport and
metabolism of a number of carbohydrates. When glucose as preferred carbon source 
is transported, EIIA(Glc) becomes predominantly unphosphorylated and
allosterically inhibits several permeases, including the maltose ATP-binding
cassette transport system (MalFGK2) in a process termed "inducer exclusion." We
have mapped the binding surface of EIIA(Glc) that interacts with the MalK
subunits by using synthetic cellulose-bound peptide arrays like pep scan- and
substitutional analyses. Three regions constituting two binding sites were
identified encompassing residues 69-79 (I), 87-91 (II), and 118-127 (III). Region
III is MalK-specific, whereas residues from regions I and II partly overlap but
are not identical to the binding interfaces for interaction with glycerol kinase 
and lactose permease. These results were fully verified by studying the
inhibitory effect of purified EIIA(Glc) variants carrying mutations at positions 
representative of each of the three regions on the ATPase activity of the
purified maltose transport complex reconstituted into proteoliposomes. Moreover, 
a synthetic peptide encompassing residues 69-91 was demonstrated to partially
inhibit ATPase activity. We also show for the first time that the N-terminal
domain of EIIA(Glc) is essential for inducer exclusion.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 16527815 [PubMed - indexed for MEDLINE]

8: J Mol Biol. 2005 Sep 30;352(4):876-92.

Crystal structures of ADP and AMPPNP-bound propionate kinase (TdcD) from
Salmonella typhimurium: comparison with members of acetate and sugar kinase/heat 
shock cognate 70/actin superfamily.

Simanshu DK, Savithri HS, Murthy MR.

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

Recently, it has been shown that l-threonine can be catabolized non-oxidatively
to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses 
the last step of this metabolic process by enabling the conversion of propionyl
phosphate and ADP to propionate and ATP. To provide insights into the
substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures 
of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have
been determined to resolutions of 2.2A and 2.3A, respectively, by molecular
replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1).
Propionate kinase, like acetate kinase, contains a fold with the topology
betabetabetaalphabetaalphabetaalpha, identical with that of glycerol kinase,
hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of
phosphotransferases. The structure consists of two domains with the active site
contained in a cleft at the domain interface. Examination of the active site
pocket revealed a plausible structural rationale for the greater specificity of
the enzyme towards propionate than acetate. This was further confirmed by kinetic
studies with the purified enzyme, which showed about ten times lower K(m) for
propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex
structures with those of acetate and sugar kinase/Hsc70/actin obtained with
different ligands has permitted the identification of catalytically essential
residues involved in substrate binding and catalysis, and points to both
structural and mechanistic similarities. In the well-characterized members of
this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large
conformational change in which the two domains close around the active site
cleft. The significant amino acid sequence similarity between TdcD and MAK has
facilitated study of domain movement, which indicates that the conformation
assumed by the two domains in the nucleotide-bound structure of TdcD may
represent an intermediate point in the pathway of domain closure.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 16139298 [PubMed - indexed for MEDLINE]

9: Exp Clin Endocrinol Diabetes. 2005 Jul;113(7):396-403.

Novel mutation (Gly280Ala) in the ATP-binding domain of glycerol kinase causes
severe hyperglycerolemia.

Wibmer T, Otto J, Parhofer KG, Otto C.

Physiological Chemistry, Adolf Butenandt Institute, University of Munich,
Germany.

Glycerol kinase deficiency is a rarely diagnosed X-linked recessive disorder
which occurs as a complex form together with the adrenal hypoplasia congenita
(AHC) or with Duchenne muscular dystrophy (DMD) or as an isolated form either
symptomatic or asymptomatic. We report the case of a male adult who had
pseudo-hypertriglyceridemia (falsely elevated triglycerides of 552 mg/dl)
refractory to lipid-lowering therapy for more than 15 years. Further
investigations revealed an isolated, asymptomatic glycerol kinase deficiency.
Using polymerase chain reaction and direct DNA sequencing, a novel missense
mutation Gly280Ala in the Xp21.3 glycerol kinase gene was found. Comparison
between human and E.coli glycerol kinase showed that the mutation affects a
highly conserved amino acid in an ATP-binding domain in the active centre. This
mutation is assumed to destabilize a hydrogen bond between ligand and enzyme
resulting in a reduced activity of glycerol kinase and therefore in
hyperglycerolemia.

Publication Types: 
    Case Reports
    Review

PMID: 16025401 [PubMed - indexed for MEDLINE]

10: Appl Environ Microbiol. 2005 Jul;71(7):4097-100.

Multiple-mutation reaction: a method for simultaneous introduction of multiple
mutations into the glpK gene of Mycoplasma pneumoniae.

Hames C, Halbedel S, Schilling O, Stülke J.

Department of General Microbiology, Institute of Microbiology and Genetics,
Georg-August University Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany.

In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a
translation stop site. This often makes it difficult to express Mycoplasma
proteins in E. coli isolates. In this work, we developed a strategy for the
one-step introduction of several mutations. This method, the multiple-mutation
reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae
glpK gene.

Publication Types: 
    Evaluation Studies
    Research Support, Non-U.S. Gov't

PMID: 16000825 [PubMed - indexed for MEDLINE]

11: Biotechnol Bioeng. 2005 Jan 20;89(2):243-51.

Estimating optimal profiles of genetic alterations using constraint-based models.

Gadkar KG, Doyle Iii FJ, Edwards JS, Mahadevan R.

Department of Chemical Engineering, University of California, Santa Barbara, CA
92121, USA.

Metabolic engineering involves application of recombinant DNA methods to
manipulate metabolic networks to improve cellular properties. It is critical that
the genetic alterations be performed in an optimal manner to maximize profit. In 
addition to the product yield, productivity consideration is also critical,
especially for the production of bulk chemicals such as 1,3-propanediol. In this 
work, we demonstrate that it is suboptimal from the standpoint of productivity to
induce genetic alteration at the start of the production process. A bi-level
optimization scheme is formulated to determine the optimal temporal flux profile 
for the manipulated reaction. In the first case study, an optimal flux in the
reaction catalyzed by glycerol kinase is determined to maximize the glycerol
production at the end of a 6-h batch cultivation of Escherichia coli under
aerobic conditions. The final glycerol concentration is 30% higher for the
optimal flux profile compared with having an active flux during the entire batch.
The effect of the mass transfer coefficient on the optimal profile and the
glycerol concentration is also determined. In the second case study, the
anaerobic batch fermentation of the ldh(-) strain of Escherichia coli is
considered. The optimal flux in the acetate pathway is determined to maximize the
final ethanol concentration. The optimal flux results in higher ethanol
concentration (11.92 mmol L(-1)) compared to strains with no acetate flux (8.36
mmol L(-1)) and fully active acetate flux (6.22 mmol L(-1)). We also examine the 
effects of growth inhibition due to high ethanol concentrations and variations in
final batch time on ethanol production.

Publication Types: 
    Comparative Study
    Evaluation Studies
    Research Support, U.S. Gov't, Non-P.H.S.

PMID: 15593263 [PubMed - indexed for MEDLINE]

12: Genome Res. 2004 Dec;14(12):2495-502.

High-throughput mutation detection underlying adaptive evolution of Escherichia
coli-K12.

Honisch C, Raghunathan A, Cantor CR, Palsson BØ, van den Boom D.

Sequenom, Inc., San Diego, California 92121, USA. chonisch@sequenom.com

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) analysis of base-specific cleavage products is an efficient,
highly accurate tool for the detection of single base sequence variations. We
describe the first application of this comparative sequencing strategy for
automated high-throughput mutation detection in microbial genomes. The method was
applied to identify DNA sequence changes that occurred in Escherichia coli K-12
MG1655 during laboratory adaptive evolution to new optimal growth phenotypes.
Experiments were based on a genome-scale in silico model of E. coli metabolism
and growth. This model computes several phenotypic functions and predicts optimal
growth rates. To identify mutations underlying a 40-d adaptive laboratory
evolution on glycerol, we resequenced 4.4% of the E. coli-K12 MG1655 genome in
several clones picked at the end of the evolutionary process. The 1.54-Mb screen 
was completed in 13.5 h. This resequencing study is the largest reported by
MALDI-TOF mass spectrometry to date. Ten mutations in 40 clones and three
deviations from the reference sequence were detected. Mutations were
predominantly found within the glycerol kinase gene. Functional characterization 
of the most prominent mutation shows its metabolic impact on the process of
adaptive evolution. All sequence changes were independently confirmed by
genotyping and Sanger-sequencing. We demonstrate that comparative sequencing by
base-specific cleavage and MALDI-TOF mass spectrometry is an automated, fast, and
highly accurate alternative to capillary sequencing.

Publication Types: 
    Comparative Study

PMID: 15574828 [PubMed - indexed for MEDLINE]

13: Biochemistry. 2004 Jan 20;43(2):362-73.

Structures of enterococcal glycerol kinase in the absence and presence of
glycerol: correlation of conformation to substrate binding and a mechanism of
activation by phosphorylation.

Yeh JI, Charrier V, Paulo J, Hou L, Darbon E, Claiborne A, Hol WG, Deutscher J.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University,
Providence, Rhode Island 02912, USA. Joanne_Yeh@brown.edu

The first structure of a glycerol kinase from a Gram-positive organism,
Enterococcus casseliflavus, has been determined to 2.8 A resolution in the
presence of glycerol and to 2.5 A resolution in the absence of substrate. The
substrate-induced closure of 7 degrees is significantly smaller than that
reported for hexokinase, a model for substrate-mediated domain closure that has
been proposed for glycerol kinase. Despite the 78% level of sequence identity and
conformational similarity in the catalytic cleft regions of the En. casseliflavus
and Escherichia coli glycerol kinases, remarkable structural differences have now
been identified. These differences correlate well with their divergent regulatory
schemes of activation by phosphorylation in En. casseliflavus and allosteric
inhibition in E. coli. On the basis of our structural results, we propose a
mechanism by which the phosphorylation of a histidyl residue located 25 A from
the active site results in a 10-15-fold increase in the activity of the
enterococcal glycerol kinase.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 14717590 [PubMed - indexed for MEDLINE]

14: Biophys J. 2003 Jul;85(1):36-48.

Mechanisms of selectivity in channels and enzymes studied with interactive
molecular dynamics.

Grayson P, Tajkhorshid E, Schulten K.

Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, 
USA.

Interactive molecular dynamics, a new modeling tool for rapid investigation of
the physical mechanisms of biological processes at the atomic level, is applied
to study selectivity and regulation of the membrane channel protein GlpF and the 
enzyme glycerol kinase. These proteins facilitate the first two steps of
Escherichia coli glycerol metabolism. Despite their different function and
architecture the proteins are found to employ common mechanisms for substrate
selectivity: an induced geometrical fit by structurally homologous binding sites 
and an induced rapid dipole moment reversal. Competition for hydrogen bonding
sites with water in both proteins is critical for substrate motion. In glycerol
kinase, it is shown that the proposed domain motion prevents competition with
water, in turn regulating the binding of glycerol.

Publication Types: 
    Comparative Study
    Evaluation Studies
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.
    Validation Studies

PMID: 12829462 [PubMed - indexed for MEDLINE]

15: Biophys J. 2003 Jul;85(1):1-4.

Engineering teams up with computer-simulation and visualization tools to probe
biomolecular mechanisms.

Schlick T.

Department of Chemistry, New York University, New York, New York 10012, USA.
schlick@nyu.edu

PMID: 12829458 [PubMed - indexed for MEDLINE]

16: Biochemistry. 2003 Apr 15;42(14):4243-52.

Linkage between fructose 1,6-bisphosphate binding and the dimer-tetramer
equilibrium of Escherichia coli glycerol kinase: critical behavior arising from
change of ligand stoichiometry.

Yu P, Pettigrew DW.

Department of Biochemistry and Biophysics, Center for Advanced Biomolecular
Research, Texas A&M University, College Station, Texas 77843-2128, USA.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase)
is inhibited allosterically by fructose 1,6-bisphosphate (FBP), and this
inhibition is a primary mechanism by which glucose controls glycerol utilization 
in vivo. Earlier work indicates that glycerol kinase displays a dimer-tetramer
equilibrium in solution, FBP shifts the equilibrium toward the tetramer, and
tetramer formation is required for FBP inhibition. However, equilibrium constants
for FBP binding and dimer-tetramer assembly that describe the linkage between
these processes are unknown. Here, decreased fluorescence anisotropy of extrinsic
fluorophores fluorescein and 2',7'-difluorofluorescein due to homo fluorescence
resonance energy transfer (homo-FRET) is used to quantitate tetramer assembly and
FBP binding. Glycerol kinase is labeled with extrinsic fluorophores covalently
attached to an engineered surface cysteine residue under conditions that prevent 
labeling of native cysteine residues. Tryptic peptide mapping and MALDI-MS verify
labeling at the engineered site only. Initial velocity studies show the labeling 
does not alter the catalytic properties or FBP inhibition. The steady-state
fluorescence anisotropy of enzyme with a labeling stoichiometry of approximately 
0.1 mol of fluorophore/mol of subunit is not sensitive to increased protein
concentration or binding of FBP, indicating the absence of homo-FRET. However,
steady-state fluorescence anisotropy of enzyme with a labeling stoichiometry of
approximately 0.4 mol of fluorophore/mol of subunit decreases with increasing
protein concentration, which is consistent with depolarization due to homo-FRET. 
The protein concentration dependence of the decreased fluorescence anisotropy is 
described by a dimer-tetramer equilibrium with an apparent dissociation constant 
of 61 +/- 7 nM (subunits) at pH 7.0 and 25 degrees C. FBP binds to both the dimer
and tetramer of glycerol kinase, and the FBP concentration dependence of the
apparent dissociation constant for the dimer-tetramer equilibrium shows critical 
behavior. The apparent dissociation constant decreases and then increases with
increasing FBP concentration, reaching a minimum at about 20 mM FBP. Critical
behavior is seen also in the FBP dependence of the inhibition. The critical
behavior arises because tetramer dissociation increases FBP stoichiometry from
two sites per tetramer to four half-sites per two dimers. The phenomenological
description of the coupling between tetramer assembly and FBP binding shows
antagonistic binding of FBP to the two sites on the tetramer, indicating that the
strong positive cooperativity observed for FBP inhibition of catalytic activity
(Hill coefficient approximately 1.5) is due to the approximately 4000-fold higher
affinity of the tetramer for FBP rather than to positive coupling between the two
FBP sites.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 12680779 [PubMed - indexed for MEDLINE]

17: DNA Seq. 2002 Dec;13(6):387-90.

Cloning and sequencing of the Thermus aquaticus glycerol facilitator gene.

Huang HS, Ito K, Yoshimoto T.

School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852, Japan.

The gene glpK, encoding glycerol kinase of Thermus aquaticus has been identified 
[Biosci. Biotechnol. Biochem., 62 (1998) 2375-2381]. In the present work, the
nucleotide sequence of glpFK operon and the gene glpF encoding glycerol
facilitator were determined. T. aquaticus GlpF was predicted to contain 272 amino
acids with six putative transmembrane segments and two half-membrane-spanning
segments that contained the motif Asn-Pro-Ala, respectively. The amino acid
residues involved in the discrimination of glycerol were deduced to be Trp44,
Tyr182, and Arg188.

PMID: 12652912 [PubMed - indexed for MEDLINE]

18: Clin Chem Lab Med. 2003 Jan;41(1):46-55.

Glycerol metabolism and the determination of triglycerides--clinical, biochemical
and molecular findings in six subjects.

Hellerud C, Burlina A, Gabelli C, Ellis JR, Nyholm PG, Lindstedt S.

Department of Clinical Chemistry, Sahlgrenska University Hospital, Göteborg
University, Gothenburg, Sweden. Christina.Hellerud@clinchem.gu.se

Recent recommendations in the National Cholesterol Education Program Expert Panel
on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults
(ATPIII) are expected to increase the number of triglyceride (TG) determinations 
and consequently the risk of misinterpretation of "non-blanked" results with
co-determination of free glycerol. Glycerol-kinase deficiency (GKD) is one cause 
of falsely elevated TG results. The natural history of isolated GKD with
symptom-free cases and cases with e.g. severe episodes of hypoglycemia and/or
ketoacidosis challenges the laboratories to identify cases of GKD and family
members at risk. "Blanked" methods reporting both glycerol and TG concentration
are therefore desirable. Molecular studies of the glycerol kinase (GK) and DAX1
genes were performed on four cases of "persistent hypertriglyceridemia" found in 
an Italian population and on two pediatric cases with high serum glycerol
concentration. Two new missense mutations were found (C358Y, T961). Molecular
modeling on GK from E. coli, indicate that these mutations are located in parts
of the enzyme important for enzyme formation or activity. One splice-site
mutation, (IVS9A-1G>A), was found in two brothers. Splice-junction analysis
indicates that it destroys the splice site and results in a mixture of mRNA.
Deletion of the GK and DAX1 genes was found in one child with symptoms of adrenal
failure. A female with glycerolemia and glyceroluria had normal GK activity but
possibly slightly decreased ability to oxidize glycerol.

Publication Types: 
    Case Reports

PMID: 12636049 [PubMed - indexed for MEDLINE]

19: Appl Environ Microbiol. 2003 Mar;69(3):1408-16.

Construction and screening of metagenomic libraries derived from enrichment
cultures: generation of a gene bank for genes conferring alcohol oxidoreductase
activity on Escherichia coli.

Knietsch A, Waschkowitz T, Bowien S, Henne A, Daniel R.

Abteilung Allgemeine Mikrobiologie, Göttingen Genomics Laboratory, Institut für
Mikrobiologie und Genetik der Georg-August-Universität, Grisebachstrasse 8, 37077
Göttingen, Germany.

Enrichment of microorganisms with special traits and the construction of
metagenomic libraries by direct cloning of environmental DNA have great potential
for identifying genes and gene products for biotechnological purposes. We have
combined these techniques to isolate novel genes conferring oxidation of
short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls.
In order to favor the growth of microorganisms containing the targeted genes,
samples collected from four different environments were incubated in the presence
of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the
four samples and used to construct complex plasmid libraries. Approximately
100,000 Escherichia coli strains of each library per test substrate were screened
for the production of carbonyls from polyols on indicator agar. Twenty-four
positive E. coli clones were obtained during the initial screen. Sixteen of them 
contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming 
phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol
oxidoreductase activity with polyols or carbonyls as the substrates in crude
extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36
complete and 17 incomplete presumptive protein-encoding genes. Fifty of these
genes showed similarity to sequenced genes from a broad collection of different
microorganisms. The genes responsible for the carbonyl formation of E. coli were 
identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110,
pAK115, and pAK116). Analyses of the amino acid sequences deduced from these
genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol
dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded
novel putative oxidoreductases belonging to groups distinct from alcohol
dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1)
encoded a protein which showed no similarity to any other known gene product.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 12620823 [PubMed - indexed for MEDLINE]

20: Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11115-20. Epub 2002 Aug 2.

Transplanting allosteric control of enzyme activity by protein-protein
interactions: coupling a regulatory site to the conserved catalytic core.

Pawlyk AC, Pettigrew DW.

Department of Biochemistry and Biophysics, Texas A&M University, College Station,
TX 77843-2128, USA.

Glycerol kinase from Escherichia coli, but not Haemophilus influenzae, is
inhibited allosterically by phosphotransferase system protein IIA(Glc). The
primary structures of these related kinases contain 501 amino acids, differing at
117. IIA(Glc) inhibition is transplanted from E. coli glycerol kinase into H.
influenzae glycerol kinase by interconverting only 11 of the differences: 8
residues that interact with IIA(Glc) at the allosteric binding site and 3
residues in the conserved ATPase catalytic core that do not interact with
IIA(Glc) but the solvent accessible surface of which decreases when it binds. The
three core residues are crucial for coupling the allosteric site to the conserved
catalytic core of the enzyme. The site of the coupling residues identifies a
regulatory locus in the sugar kinase/heat shock protein 70/actin superfamily and 
suggests relations between allosteric regulation and the active site closure that
characterizes the family. The location of the coupling residues provides
empirical validation of a computational model that predicts a coupling pathway
between the IIA(Glc)-binding site and the active site [Luque, I. & Freire, E.
(2000) Proteins Struct. Funct. Genet. Suppl. 4, 63-71]. The requirement for
changes in core residues to couple the allosteric and active sites and switching 
from inhibition to activation by a single amino acid change are consistent with a
postulated mechanism for molecular evolution of allosteric regulation.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 12161559 [PubMed - indexed for MEDLINE]

21: Biotechnol Prog. 2002 Jul-Aug;18(4):694-9.

Improving 1,3-propanediol production from glycerol in a metabolically engineered 
Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate.

Zhu MM, Lawman PD, Cameron DC.

Department of Chemical Engineering, University of Wisconsin-Madison, Madison,
Wisconsin 53706-1691. marie_zhu@groton.pfizer.com

High levels of glycerol significantly inhibit cell growth and 1,3-propanediol
(1,3-PD) production in anaerobic glycerol fermentation by genetically engineered 
Escherichia coli (E. coli) strains expressing genes from the Klebsiella
pneumoniae dha (K.pneumoniae) regulon. We have previously demonstrated that
1,3-PD production by the engineered E. coli can be improved by reducing the
accumulation of methylglyoxal. This study focuses on investigation of another
lesser-known metabolite in the pathways related to 1,3-PD
production-glycerol-3-phosphate (G3P). When grown anaerobically on glycerol in
the absence of an exogenous acceptor, the engineered E. coli strains have
intracellular G3P levels that are significantly higher than those in K.
pneumoniae, a natural 1,3-PD producer. Furthermore, in the engineered E. coli
strains, the G3P levels increase with increasing glycerol concentrations,
whereas, in K. pneumoniae, the concentrations of G3P remain relatively constant. 
Addition of fumarate, which can stimulate activity of anaerobic G3P
dehydrogenase, into the fermentation medium led to a greater than 30-fold
increase in the specific activity of anaerobic G3P dehydrogenase and a
significant decrease in concentrations of intracellular G3P and resulted in
better cell growth and an improved production of 1,3-PD. This indicates that the 
low activity of G3P dehydrogenase in the absence of an exogenous electron
acceptor is one of the reasons for G3P accumulation. In addition, spent media
from E.coli Lin61, a glycerol kinase (responsible for conversion of glycerol to
G3P) mutant, contains greatly decreased concentrations of G3P and shows improved 
production of 1,3-PD (by 2.5-fold), when compared to media from its parent strain
E. coli K10. This further suggests that G3P accumulation is one of the reasons
for the inhibition of 1,3-PD production during anaerobic fermentation.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 12153300 [PubMed - indexed for MEDLINE]

22: Free Radic Res. 2001 Dec;35(6):867-72.

Glycerol metabolism in superoxide dismutase-deficient Escherichia coli.

Benov L, Al-Ibraheem J.

Department of Biochemistry, Faculty of Medicine, Kuwait University, Safat.
lbenov@hsc.kuniv.edu.kw

Escherichia coli, which lacks cytoplasmic superoxide dismutases, exhibits various
phenotypic deficits if grown aerobically. Here we report that sodAsodB E. coli
cannot use glycerol under aerobic conditions. The reason is low activity of
glycerol kinase (GK), the rate-limiting enzyme in glycerol metabolism. Superoxide
does not inactivate GK, but makes it susceptible to inactivation by a heat-labile
factor present in the cell-free extracts. This factor seems to be part of a
proteolytic system, which recognizes and degrades oxidatively modified proteins.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 11811537 [PubMed - indexed for MEDLINE]

23: J Biol Chem. 2002 Jan 25;277(4):2682-6. Epub 2001 Nov 19.

Effects of modulation of glycerol kinase expression on lipid and carbohydrate
metabolism in human muscle cells.

Montell E, Lerín C, Newgard CB, Gómez-Foix AM.

Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona, Marti i
Franquès, 1, 08028 Barcelona, Spain.

Glycerol is taken up by human muscle in vivo and incorporated into lipids, but
little is known about regulation of glycerol metabolism in this tissue. In this
study, we have analyzed the role of glycerol kinase (GlK) in the regulation of
glycerol metabolism in primary cultured human muscle cells. Isolated human muscle
cells exhibited lower GlK activity than fresh muscle explants, but the activity
in cultured cells was increased by exposure to insulin. [U-(14)C]Glycerol was
incorporated into cellular phospholipids and triacylglycerides (TAGs), but little
or no increase in TAG content or lactate release was observed in response to
changes in the medium glycerol concentration. Adenovirus-mediated delivery of the
Escherichia coli GlK gene (AdCMV-GlK) into muscle cells caused a 30-fold increase
in GlK activity, which was associated with a marked rise in the labeling of
phospholipid or TAG from [U-(14)C]glycerol compared with controls. Moreover, GlK 
overexpression caused [U-(14)C]glycerol to be incorporated into glycogen, which
was dependent on the activation of glycogen synthase. Co-incubation of
AdCMV-GlK-treated muscle cells with glycerol and oleate resulted in a large
accumulation of TAG and an increase in lactate production. We conclude that GlK
is the limiting step in muscle cell glycerol metabolism. Glycerol 3-phosphate is 
readily used for TAG synthesis but can also be diverted to form glycolytic
intermediates that are in turn converted to glycogen or lactate. Given the high
levels of glycerol in muscle interstitial fluid, these finding suggest that
changes in GlK activity in muscle can exert important influences on fuel
deposition in this tissue.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 11714702 [PubMed - indexed for MEDLINE]

24: Biochemistry. 2001 Nov 27;40(47):14302-8.

IIA(Glc) allosteric control of Escherichia coli glycerol kinase: binding site
cooperative transitions and cation-promoted association by Zinc(II).

Holtman CK, Pawlyk AC, Meadow N, Roseman S, Pettigrew DW.

Department of Biochemistry and Biophysics, Center for Advanced Biomolecular
Research, and Program in Microbial Genetics and Genomics, Texas A&M University,
College Station, Texas 77843-2128, USA.

The catalytic activity of glycerol kinase (EC 2.7.1.30, ATP:glycerol
3-phosphotransferase) from Escherichia coli is inhibited allosterically by
IIA(Glc) (previously known as III(Glc)), the glucose-specific phosphocarrier
protein of the phosphoenolpyruvate:glycose phosphotransferase system. A
sequentially contiguous portion of glycerol kinase undergoes an induced fit
conformational change involving coil, alpha-helix, and 3(10)-helix upon IIA(Glc) 
binding. A second induced fit occurs upon binding of Zn(II) to a novel
intermolecular site, which increases complex stability by cation-promoted
association. Eight of the ten sequentially contiguous amino acids are substituted
with alanine to evaluate the roles of these positions in complex formation.
Effects of the substitutions reveal both favorable and antagonistic contributions
of the normal amino acids to complex formation, and Zn(II) reverses these
contributions for two of the amino acids. The consequences of some of the
substitutions for IIA(Glc) inhibition are consistent with changes in the
intermolecular interactions seen in the crystal structures. However, for the
amino acids that are located in the region that is alpha-helical in the absence
of IIA(Glc), the effects of the substitutions are not consistent with changes in 
intermolecular interactions but with increased stability of the alpha-helical
region due to the higher alpha-helix propensity of alanine. The reduced affinity 
for IIA(Glc) binding seen for these variants is consistent with predictions of
Freire and co-workers [Luque, I., and Freire, E. (2000) Proteins: Struct.,
Funct., Genet. 4, 63-71]. These variants show also increased cation-promoted
association by Zn(II) so that the energetic contribution of Zn(II) to complex
formation is doubled. The similarity of effects of the alanine substitutions of
the amino acids in the alpha-helical region for IIA(Glc) binding affinity and
cation-promoted association by Zn(II) indicates that they function as a
cooperative unit.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 11714284 [PubMed - indexed for MEDLINE]

25: J Bacteriol. 2001 Oct;183(19):5675-83.

External-pH-dependent expression of the maltose regulon and ompF gene in
Escherichia coli is affected by the level of glycerol kinase, encoded by glpK.

Chagneau C, Heyde M, Alonso S, Portalier R, Laloi P.

Unité de Microbiologie et Génétique, UMR CNRS 5122, Université Lyon 1, F-69622
Villeurbanne Cedex, France.

The expression of the maltose system in Escherichia coli is regulated at both
transcriptional and translational levels by the pH of the growth medium (pHo).
With glycerol as the carbon source, transcription of malT, encoding the
transcriptional activator of the maltose regulon, is weaker in acidic medium than
in alkaline medium. malT transcription became high, regardless of the pHo, when
glycerol-3-phosphate or succinate was used as the carbon source. Conversely, malT
expression was low, regardless of the pHo, when maltose was used as the carbon
source. The increase in malT transcription, associated with the pHo, requires the
presence of glycerol in the growth medium and the expression of the glycerol
kinase (GlpK). Changes in the level of glpK transcription had a great effect on
malT transcription. Indeed, a glpFKX promoter-down mutation has been isolated,
and in the presence of this mutation, malT expression was increased. When glpK
was expressed from a high-copy-number plasmid, the glpK-dependent reduced
expression of the maltose system became effective regardless of the pHo. Analysis
of this repression showed that a malTp1 malTp10 promoter, which is independent of
the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex, was no longer
repressed by glpFKX amplification. Thus, GlpK-dependent repression of the maltose
system requires the cAMP-CRP complex. We propose that the pHo may affect a
complex interplay between GlpK, the phosphotransferase-mediated uptake of
glucose, and the adenylate cyclase.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 11544231 [PubMed - indexed for MEDLINE]

26: J Bacteriol. 2001 Aug;183(15):4493-8.

Properties of a revertant of Escherichia coli viable in the presence of
spermidine accumulation: increase in L-glycerol 3-phosphate.

Raj VS, Tomitori H, Yoshida M, Apirakaramwong A, Kashiwagi K, Takio K, Ishihama
A, Igarashi K.

Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho,
Inage-ku, Chiba 263-8522, Japan.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine
acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM 
spermidine, their viability was greatly decreased through the inhibition of
protein synthesis by overaccumulation of spermidine. When the cells were cultured
with a high concentration of spermidine (4 mM), a revertant strain was obtained. 
We found that a 55-kDa protein, glycerol kinase, was overexpressed in the
revertant and that synthesis of a ribosome modulation factor and the RNA
polymerase sigma(38) subunit, factors important for cell viability, was increased
in the revertant. Levels of L-glycerol 3-phosphate also increased in the
revertant. Transformation of glpFK, which encodes a glycerol diffusion
facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially
prevented the cell death caused by accumulation of spermidine. It was also found 
that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and
attenuated the inhibition of protein synthesis caused by high concentrations of
spermidine. These results indicate that L-glycerol 3-phosphate reduces the
binding of excess amounts of spermidine to ribosomes so that protein synthesis is
recovered.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 11443083 [PubMed - indexed for MEDLINE]

27: Protein Expr Purif. 2001 Jun;22(1):52-9.

Subcloning, expression, purification, and characterization of Haemophilus
influenzae glycerol kinase.

Pawlyk AC, Pettigrew DW.

Department of Biochemistry and Biophysics, Program in Microbial Genetics and
Genomics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA.

Glycerol kinase (EC 2.7.1.30) is a bacterial sugar kinase and a member of the
sugar kinase/actin/hsc-70 superfamily of enzymes. The enzyme from Escherichia
coli is an allosteric regulatory enzyme whose activity is inhibited by fructose
1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier of the
phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (previously
termed III(Glc)). Comparison of its primary structure with that of the highly
similar Haemophilus influenzae glycerol kinase reveals that the amino acid
sequence for the binding site for FBP is conserved while the amino acid sequence 
for the binding site for IIA(Glc) contains differences that are predicted to
prevent its inhibition. To test this hypothesis, the H. influenzae glpK gene was 
assembled from DNA library fragments and subcloned into pUC18. The enzyme is
expressed at high levels in E. coli. It was purified to greater than 90%
homogeneity by taking advantage of its solubility behavior in a procedure that
requires no column chromatography. The initial-velocity kinetic parameters of the
purified enzyme are similar to those of the E. coli glycerol kinase. The H.
influenzae glycerol kinase is inhibited by FBP but not by IIA(Glc), in agreement 
with the prediction based on sequence comparison. Sedimentation velocity
experiments reveal that inhibition of HiGK by FBP is associated with
oligomerization, behavior which is similar to EcGK. The possibility of utilizing 
mutagenesis studies to exploit the high degree of similarity of these two enzymes
to elucidate the mechanism of allosteric regulation by IIA(Glc) is discussed.
Copyright 2001 Academic Press.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 11388799 [PubMed - indexed for MEDLINE]

28: J Mol Microbiol Biotechnol. 2001 Jul;3(3):347-54.

Three-dimensional structures of protein-protein complexes in the E. coli PTS.

Peterkofsky A, Wang G, Garrett DS, Lee BR, Seok YJ, Clore GM.

Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute,
National Institutes of Health, Bethesda, MD 20892-4036, USA. alan@codon.nih.gov

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes 
a collection of proteins that accomplish phosphoryl transfer from
phosphoenolpyruvate (PEP) to a sugar in the course of transport. The soluble
proteins of the glucose transport pathway also function as regulators of diverse 
systems. The mechanism of interaction of the phosphoryl carrier proteins with
each other as well as with their regulation targets has been amenable to study by
nuclear magnetic resonance (NMR) spectroscopy. The three-dimensional solution
structures of the complexes between the N-terminal domain of enzyme I and HPr and
between HPr and enzyme IIA(Glc) have been elucidated. An analysis of the binding 
interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed
that a common surface on HPr is involved in all these interactions. Similarly, a 
common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase.
Thus, there is a common motif for the protein-protein interactions characteristic
of the PTS.

Publication Types: 
    Review

PMID: 11361064 [PubMed - indexed for MEDLINE]

29: J Bacteriol. 2001 Jun;183(11):3336-44.

Reverse genetics of Escherichia coli glycerol kinase allosteric regulation and
glucose control of glycerol utilization in vivo.

Holtman CK, Pawlyk AC, Meadow ND, Pettigrew DW.

Department of Biochemistry and Biophysics, Program in Microbial Genetics and
Genomics, Texas A&M University, College Station, TX 77843-2128, USA.

Reverse genetics is used to evaluate the roles in vivo of allosteric regulation
of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the
phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (formerly known
as III(glc)), and by fructose 1,6-bisphosphate. Roles have been postulated for
these allosteric effectors in glucose control of both glycerol utilization and
expression of the glpK gene. Genetics methods based on homologous recombination
are used to place glpK alleles with known specific mutations into the chromosomal
context of the glpK gene in three different genetic backgrounds. The alleles
encode glycerol kinases with normal catalytic properties and specific alterations
of allosteric regulatory properties, as determined by in vitro characterization
of the purified enzymes. The E. coli strains with these alleles display the
glycerol kinase regulatory phenotypes that are expected on the basis of the in
vitro characterizations. Strains with different glpR alleles are used to assess
the relationships between allosteric regulation of glycerol kinase and specific
repression in glucose control of the expression of the glpK gene. Results of
these studies show that glucose control of glycerol utilization and glycerol
kinase expression is not affected by the loss of IIA(Glc) inhibition of glycerol 
kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is
the dominant allosteric control mechanism, and glucose is unable to control
glycerol utilization in its absence. Specific repression is not required for
glucose control of glycerol utilization, and the relative roles of various
mechanisms for glucose control (catabolite repression, specific repression, and
inducer exclusion) are different for glycerol utilization than for lactose
utilization.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 11344141 [PubMed - indexed for MEDLINE]

30: J Bacteriol. 2001 Feb;183(4):1459-61.

Unexpected presence of defective glpR alleles in various strains of Escherichia
coli.

Holtman CK, Thurlkill R, Pettigrew DW.

Department of Biochemistry and Biophysics, Texas A&M University, College Station,
Texas 77843-2128, USA.

Alleles of glpR associated with the same GlpR(-) phenotype produce substitutions 
in different conserved portions of the glycerol 3-phosphate repressor which are
not part of the helix-turn-helix motif. Analysis of the effects on growth and
enzyme expression show that glucose repression of glycerol utilization is not
dependent on a functional repressor.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 11157961 [PubMed - indexed for MEDLINE]

31: Biol Chem. 2000 Nov;381(11):1071-7.

Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLK1)
from Trypanosoma brucei.

Steinborn K, Szallies A, Mecke D, Duszenko M.

Physiologisch-chemisches Institut, Universität Tübingen, Germany.

We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma
brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and
contains an ORF encoding a protein with high homology to known glycerol kinases
of other organisms. It is 512 amino acids in length with a PTS1-like targeting
sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of
this enzyme. Although Northern blot analysis revealed higher mRNA levels in
slender bloodstream forms than in the procyclic insect forms, specific glycerol
kinase activities were found to be virtually identical in both life stages.
Southern blot analysis suggested a single copy gene, but we were able to clone
two alleles utmost similar to each other. Heterologous expression of the
trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis 
of this enzyme. In particular, we have been able to monitor ATP production from
glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very
unfavorable, is regarded essential for the survival of Trypanosoma brucei under
anoxic conditions. Since the unique spatial separation of glycolysis in the
kinetoplastida imposes important consequences for the regulation of the energy
metabolism in these organisms, we discuss the observed differences between TbGLK1
and glycerol kinases from other organisms in view of its physiological relevance.

PMID: 11154065 [PubMed - indexed for MEDLINE]

32: EMBO J. 2000 Nov 1;19(21):5635-49.

Solution structure of the phosphoryl transfer complex between the signal
transducing proteins HPr and IIA(glucose) of the Escherichia coli
phosphoenolpyruvate:sugar phosphotransferase system.

Wang G, Louis JM, Sondej M, Seok YJ, Peterkofsky A, Clore GM.

Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
20892-0510, USA.

The solution structure of the second protein-protein complex of the Escherichia
coli phosphoenolpyruvate: sugar phosphotransferase system, that between
histidine-containing phosphocarrier protein (HPr) and glucose-specific enzyme
IIA(Glucose) (IIA(Glc)), has been determined by NMR spectroscopy, including the
use of dipolar couplings to provide long-range orientational information and
newly developed rigid body minimization and constrained/restrained simulated
annealing methods. A protruding convex surface on HPr interacts with a
complementary concave depression on IIA(Glc). Both binding surfaces comprise a
central hydrophobic core region surrounded by a ring of polar and charged
residues, positive for HPr and negative for IIA(Glc). Formation of the
unphosphorylated complex, as well as the phosphorylated transition state,
involves little or no change in the protein backbones, but there are
conformational rearrangements of the interfacial side chains. Both HPr and
IIA(Glc) recognize a variety of structurally diverse proteins. Comparisons with
the structures of the enzyme I-HPr and IIA(Glc)-glycerol kinase complexes reveal 
how similar binding surfaces can be formed with underlying backbone scaffolds
that are structurally dissimilar and highlight the role of redundancy and side
chain conformational plasticity.

Publication Types: 
    Comparative Study
    Research Support, U.S. Gov't, P.H.S.

PMID: 11060015 [PubMed - indexed for MEDLINE]

33: Biochemistry. 2000 Oct 10;39(40):12303-11.

Secondary structure and oligomerization of the E. coli glycerol facilitator.

Manley DM, McComb ME, Perreault H, Donald LJ, Duckworth HW, O'Neil JD.

Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2,
Canada.

The Major Intrinsic Proteins are found throughout the bacterial, plant, and
animal kingdoms and are responsible for the rapid transport of water and other
small, polar solutes across membranes. The superfamily includes the aquaporins,
the aquaglyceroporins, and the glycerol facilitators. We have overexpressed and
purified the Escherichia coli inner membrane glycerol facilitator. Approximately 
7.5 mg of 95% pure protein is obtained from 1 L of Escherichia coli cells using
immobilized metal affinity chromatography. Well-resolved matrix-assisted laser
desorption ionization mass spectra were obtained by solubilization of the protein
in octyl-beta-D-glucopyranoside (M(r) = 33 650.3; error approximately 0.4%). The 
recombinant glycerol facilitator is inserted into the bacterial inner membrane,
is functional, and is inhibited by HgCl(2). Polyacrylamide gel electrophoresis
suggests that the facilitator is predominantly monomeric when solubilized with
dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, and sodium dodecyl
sulfate, but that it self-associates, forming soluble oligomers when urea is used
during extraction. Similar oligomeric species are demonstrated to exist in the
bacterial membrane by chemical cross-linking experiments. Circular dichroism
analysis shows that the protein is predominantly alpha-helical. Helix content is 
significantly higher in protein prepared in the absence of urea (42-55%) than in 
its presence (32%). A possible role for the facilitator oligomers in interactions
with, and regulation of, the glycerol kinase is discussed.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 11015209 [PubMed - indexed for MEDLINE]

34: J Bacteriol. 2000 Oct;182(19):5624-7.

Purification and characterization of glpX-encoded fructose 1, 6-bisphosphatase, a
new enzyme of the glycerol 3-phosphate regulon of Escherichia coli.

Donahue JL, Bownas JL, Niehaus WG, Larson TJ.

Department of Biochemistry, Virginia Polytechnic Institute and State University, 
Blacksburg, Virginia 24061, USA.

In Escherichia coli, gene products of the glp regulon mediate utilization of
glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol
diffusion facilitator, glycerol kinase, and as shown here, a fructose
1,6-bisphosphatase that is distinct from the previously described fbp-encoded
enzyme. The purified enzyme was dimeric, dependent on Mn(2+) for activity, and
exhibited an apparent K(m) of 35 microM for fructose 1,6-bisphosphate. The enzyme
was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.

Publication Types: 
    Research Support, U.S. Gov't, Non-P.H.S.

PMID: 10986273 [PubMed - indexed for MEDLINE]

35: Eur J Biochem. 2000 Apr;267(8):2323-33.

Glycerol kinase of Trypanosoma brucei. Cloning, molecular characterization and
mutagenesis.

Králová I, Rigden DJ, Opperdoes FR, Michels PA.

Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular
Pathology and Laboratory of Biochemistry, Université Catholique de Louvain,
Brussels, Belgium.

Trypanosoma brucei contains two tandemly arranged genes for glycerol kinase. The 
downstream gene was analysed in detail. It contains an ORF for a polypeptide of
512 amino acids. The polypeptide has a calculated molecular mass of 56 363 Da and
a pI of 8.6. Comparison of the T. brucei glycerol kinase amino-acid sequence with
the glycerol kinase sequences available in databases revealed positional
identities of 39.0-50.4%. The T. brucei glycerol kinase gene was overexpressed in
Escherichia coli cells and the recombinant protein obtained was purified and
characterized biochemically. Its kinetic properties with regard to both the
forward and reverse reaction were measured. The values corresponded to those
determined previously for the natural glycerol kinase purified from the parasite,
and confirmed that the apparent Km values of the trypanosome enzyme for its
substrates are relatively high compared with those of other glycerol kinases.
Alignment of the amino-acid sequences of T. brucei glycerol kinase and other
eukaryotic and prokaryotic glycerol kinases, as well as inspection of the
available three-dimensional structure of E. coli glycerol kinase showed that most
residues of the magnesium-, glycerol- and ADP-binding sites are well conserved in
T. brucei glycerol kinase. However, a number of remarkable substitutions was
identified, which could be responsible for the low affinity for the substrates.
Most striking is amino-acid Ala137 in T. brucei glycerol kinase; in all other
organisms a serine is present at the corresponding position. We mutated Ala137 of
T. brucei glycerol kinase into a serine and this mutant glycerol kinase was
over-expressed and purified. The affinity of the mutant enzyme for its substrates
glycerol and glycerol 3-phosphate appeared to be 3. 1-fold to 3.6-fold higher
than in the wild-type enzyme. Part of the glycerol kinase gene comprising this
residue 137 was amplified in eight different kinetoplastid species and sequenced.
Interestingly, an alanine occurs not only in T. brucei, but also in other
trypanosomatids which can convert glucose into equimolar amounts of glycerol and 
pyruvate: T. gambiense, T. equiperdum and T. evansi. In trypanosomatids with no
or only a limited capacity to produce glycerol, a hydroxy group-containing
residue is found as in all other organisms: T. vivax and T. congolense possess a 
serine while Phytomonas sp., Leishmania brasiliensis and L. mexicana have a
threonine.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 10759857 [PubMed - indexed for MEDLINE]

36: Ann N Y Acad Sci. 1999 May 18;870:383-5.

Specificity of transcription-enhanced mutations.

Longacre A, Reimers JM, Wright BE.

Division of Biological Sciences, University of Montana, Missoula 59812, USA.

PMID: 10415503 [PubMed - indexed for MEDLINE]

37: Biochem Biophys Res Commun. 1999 Jun 16;259(3):640-4.

X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel
mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase.

Mao C, Ozer Z, Zhou M, Uckun FM.

Drug Discovery Program, Hughes Institute, St. Paul, Minnesota, 55113, USA.
cmao@mercury.ih.org

Glycerol kinase (GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol
which yields glycerol 3-phosphate. The 2.8 A new crystal structure of GK
complexed with an ATP analog revealed an unexpected position of the
gamma-phosphoryl group, which was 7.2 A distant from the 3-hydroxyl group of
glycerol, 5.5 A away from the 3-phosphate of the product (glycerol 3-phosphate)
and is stabilized by a beta-hairpin structure. Based on the presented crystal
structure and the previously determined structures of GK product complexes, we
propose a 3-D model of a nucleophilic in-line transfer mechanism for the
ATP-dependent phosphorylation of glycerol by GK. Copyright 1999 Academic Press.

PMID: 10364471 [PubMed - indexed for MEDLINE]

38: Biochemistry. 1999 Mar 23;38(12):3508-18.

Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex
with nonhydrolyzable ATP analogues reveal a putative active conformation of the
enzyme as a result of domain motion.

Bystrom CE, Pettigrew DW, Branchaud BP, O'Brien P, Remington SJ.

Institute of Molecular Biology, Departments of Physics and Chemistry, University 
of Oregon, Eugene, Oregon 97403, USA.

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity
toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which
has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. 
To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to 
sterically block formation of the dimer-dimer interface near the FBP binding site
[Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37,
16565-16572]. The substitution did not substantially change the Michaelis
constants or alter allosteric regulation of GK by a second effector, the
phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal
structures of GK in complex with different nontransferable ATP analogues and
glycerol revealed an asymmetric dimer with one subunit adopting an open
conformation and the other adopting the closed conformation found in previously
determined structures. The conformational difference is produced by a
approximately 6.0 degrees rigid-body rotation of the N-terminal domain with
respect to the C-terminal domain, similar to that observed for hexokinase and
actin, members of the same ATPase superfamily. Two of the ATP analogues bound in 
nonproductive conformations in both subunits. However, beta,
gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor 
of GK, bound nonproductively in the closed subunit and in a putative productive
conformation in the open subunit, with the gamma-phosphate placed for in-line
transfer to glycerol. This asymmetry is consistent with "half-of-the-sites"
reactivity and suggests that the inhibition of GK by FBP is due to restriction of
domain motion.

Publication Types: 
    Comparative Study
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 10090737 [PubMed - indexed for MEDLINE]

39: Biosci Biotechnol Biochem. 1998 Dec;62(12):2388-95.

A novel glycerol kinase from Flavobacterium meningosepticum: characterization,
gene cloning and primary structure.

Sakasegawa S, Yoshioka I, Koga S, Takahashi M, Matsumoto K, Misaki H, Ohshima T.

Asahi Chemical Industry Co. Ltd., Shizuoka, Japan. a9310932@ut.asahi-kasei.co.jp

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from
Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa
by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 
and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and 
at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli
and its complete DNA was sequenced. The FGK gene consists of an open reading
frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid
sequence of the gene had 40-60% similarity to those of glycerol kinases from
other origins and the amino acid sequence of the putative active site residue
reported for E. coli GK is identical to the corresponding sequence of FGK except 
for one amino acid residue.

PMID: 9972265 [PubMed - indexed for MEDLINE]

40: Biosci Biotechnol Biochem. 1998 Dec;62(12):2375-81.

Cloning, sequencing, high expression, and crystallization of the thermophile
Thermus aquaticus glycerol kinase.

Huang HS, Ito K, Yin CH, Kabashima T, Yoshimoto T.

School of Pharmaceutical Sciences, Nagasaki University, Japan.

Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism
in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK,
from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a
protein of 496 amino acids with a predicted molecular weight of 54,814. The amino
acid sequence deduced from T. aquaticus glpK was found to have identities of 97
and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK
genes. After overproduction in Escherichia coli, the expressed enzyme was easily 
purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has
been crystallized by the hanging drop vapor diffusion method at 22 degrees C.
Comparison of the amino acid sequence with that of the B. subtilis enzyme showed 
that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and
thermophile enzymes.

PMID: 9972264 [PubMed - indexed for MEDLINE]

41: Protein Eng. 1998 Dec;11(12):1219-27.

Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and 
characterization of the recombinant enzyme.

Koga Y, Morikawa M, Haruki M, Nakamura H, Imanaka T, Kanaya S.

Department of Material and Life Science, Graduate School of Engineering, Osaka
University, Suita, Japan.

The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic
archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia
coli. The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide 
sequence showed 57% identity with that of E. coli GK and 47% identity with that
of human GK. Pk-GK, which has a molecular weight of 55902 (497 amino acid
residues), was purified from E. coli and characterized. Despite the high sequence
similarity, Pk-GK and E. coli GK are greatly divergent in structure and function 
from each other. Unlike E. coli GK, which exists as a tetramer, Pk-GK exists as a
dimer. The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+. The
optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C,
respectively. Pk-GK can utilize other nucleoside triphosphates than ATP as a
phosphoryl donor. It is fairly resistant to an allosteric inhibitor of E. coli
GK, fructose-1,6-bisphosphate. Determination of the kinetic parameters indicates 
that the Km value of the enzyme is 15.4 microM for ATP and 111 microM for
glycerol and its kcat value is 940 s(-1). The enzyme was shown to be fairly
resistant to irreversible heat inactivation and still retained 50% of its
enzymatic activity even after heating at 100 degrees C for 30 min. Construction
of a model for the three-dimensional structure of the enzyme suggests that the
formation of extensive ion-pair networks is responsible for the high stability of
this enzyme.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't

PMID: 9930671 [PubMed - indexed for MEDLINE]

42: J Bacteriol. 1999 Jan;181(2):632-41.

Phosphorylation and functional properties of the IIA domain of the lactose
transport protein of Streptococcus thermophilus.

Gunnewijk MG, Postma PW, Poolman B.

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, 9751 NN Haren, The Netherlands.

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a
carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of
proteins and protein domains of the phosphoenolpyruvate:carbohydrate
phosphotransferase system (PTS) in various organisms, of which IIAGlc of
Escherichia coli is the best-characterized member. On the basis of these
similarities, it was anticipated that IIALacS would be able to perform one or
more functions associated with IIAGlc, i.e., carry out phosphoryl transfer and/or
affect other catabolic functions. The gene fragment encoding IIALacS was
overexpressed in Escherichia coli, and the protein was purified in two steps by
metal affinity and anion-exchange chromatography. IIALacS was unable to restore
glucose uptake in a IIAGlc-deficient strain, which is consistent with a very low 
rate of phosphorylation of IIALacS by phosphorylated HPr (HPr approximately P)
from E. coli. With HPr approximately P from S. thermophilus, the rate was more
than 10-fold higher, but the rate constants for the phosphorylation of IIALacS
(k1 = 4.3 x 10(2) M-1 s-1) and dephosphorylation of IIALacS approximately P by
HPr (k-1 = 1.1 x 10(3) M-1 s-1) are still at least 4 orders of magnitude lower
than for the phosphoryltransfer between IIAGlc and HPr from E. coli. This finding
suggests that IIALacS has evolved into a protein domain whose main function is
not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of 
IIA proteins with and without putative phosphoryl transfer functions and the
known structure of IIAGlc, we constructed a double mutant [IIALacS(I548E/G556D)] 
that was predicted to have increased phosphoryl transfer activity. Indeed, the
phosphorylation rate of IIALacS(I548E/G556D) by HPr approximately P increased (k1
= 4.0 x 10(3) M-1 s-1) and became nearly independent of the source of HPr
approximately P (S. thermophilus, Bacillus subtilis, or E. coli). The increased
phosphoryl transfer rate of IIALacS(I548E/G556D) was insufficient to complement
IIAGlc in PTS-mediated glucose transport in E. coli. Both IIALacS and
IIALacS(I548E/G556D) could replace IIAGlc, but in another function: they
inhibited glycerol kinase (inducer exclusion) when present in the
unphosphorylated form.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 9882680 [PubMed - indexed for MEDLINE]

43: Biochemistry. 1998 Nov 24;37(47):16565-72.

Crystal structure of a complex of Escherichia coli glycerol kinase and an
allosteric effector fructose 1,6-bisphosphate.

Ormö M, Bystrom CE, Remington SJ.

Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.

The three-dimensional structures of Escherichia coli glycerol kinase (GK) with
bound glycerol in the presence and absence of one of the allosteric regulators of
its activity, fructose 1,6-bisphosphate (FBP), at 3.2 and 3.0 A, are presented.
The molecule crystallized in space group P41212, and the structure was solved by 
molecular replacement. The models were refined with good stereochemistry to final
R-factors of 21.1 and 21.9%, respectively. A tetrameric arrangement of monomers
was observed which was essentially identical to the proposed inactive tetramer II
previously described [Feese, M. D., Faber, H. R., Bystrom, C. E., Pettigrew, D.
W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal
packing in this form was especially open, permitting the FBP binding site to be
occupied and identified. The crystallographic data revealed a most unusual type
of FBP binding site formed between two glycine-arginine loops (residues 234-236) 
where one-half of the binding site is donated by each monomer at the regulatory
interface. The molecule of FBP binds in two mutually exclusive modes on a
noncrystallographic 2-fold axis at 50% occupancy each; thus, a tetramer of GK
binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of
FBP and Arg 236 were observed in addition to hydrogen bonding interactions
between the backbone amide of Gly 234 and the 6-phosphate. No contacts between
the protein and the furanose ring were observed. Mutagenesis of Arg 236 to
alanine drastically reduced the extent of inhibition of GK by FBP and lowered,
but did not eliminate, the ability of FBP to promote tetramer association. These 
observations are consistent with the previously characterized mechanism of FBP
inhibition of GK, in which FBP acts both to promote dimer-tetramer assembly and
to inactivate the tetramers.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 9843423 [PubMed - indexed for MEDLINE]

44: Structure. 1998 Nov 15;6(11):1407-18.

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal
structures reveal conformational changes with implications for allosteric
regulation.

Feese MD, Faber HR, Bystrom CE, Pettigrew DW, Remington SJ.

Central Laboratories for Key Technology 1 - 13-5 Fukuura Kanazawa Yokohama 236,
Japan.

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V
system) enzyme that has three allosteric effectors with independent mechanisms:
fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine
nucleotides. The enzyme exists in solution as functional dimers that associate
reversibly to form tetramers. GK is a member of a superfamily of ATPases that
share a common ATPase domain and are thought to undergo a large conformational
change as an intrinsic step in their catalytic cycle. Members of this family
include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report
here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex
with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric
tetramer. The functional dimer is identical to that described previously for the 
IIAGlc-GK complex structure. The tetramer interface is significantly different,
however, with a relative 22.3 degrees rotation and 6.34 A translation of one
functional dimer. The overall monomer structure is unchanged except for two
regions: the IIAGlc-binding site undergoes a structural rearrangement and
residues 230-236 become ordered and bind orthophosphate at the tetramer
interface. We also report the structure of a second mutant of GK (IIe474-->Asp)
in complex with IIAGlc; this complex crystallized isomorphously to the wild type 
IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the
IIAGlc-binding site show significantly altered kinetic and regulatory properties,
suggesting that the conformation of the binding site is linked to the regulation 
of activity. CONCLUSIONS: We conclude that the new tetramer structure presented
here is an inactive form of the physiologically relevant tetramer. The structure 
and location of the orthophosphate-binding site is consistent with it being part 
of the FBP-binding site. Mutational analysis and the structure of the
IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the
IIAGlc-binding site to be an essential aspect of IIAGlc regulation.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 9817843 [PubMed - indexed for MEDLINE]

45: Biochemistry. 1998 Apr 7;37(14):4875-83.

Cation-promoted association of Escherichia coli phosphocarrier protein IIAGlc
with regulatory target protein glycerol kinase: substitutions of a Zinc(II)
ligand and implications for inducer exclusion.

Pettigrew DW, Meadow ND, Roseman S, Remington SJ.

Department of Biochemistry & Biophysics, Texas A&M University, College Station,
Texas 77843-2128, USA.

In Escherichia coli, inducer exclusion is one mechanism by which glucose prevents
unnecessary expression of genes needed for metabolism of other sugars. The basis 
for this mechanism is binding of the unphosphorylated form of the
glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose
phosphotransferase system, IIAGlc (also known as IIIGlc), to a variety of target 
proteins to prevent uptake or synthesis of the inducer. One of these target
proteins is glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase),
which is inhibited by IIAGlc. Glycerol kinase is the only IIAGlc target protein
for which the structure of the complex is known. Association of these two
proteins forms an intermolecular binding site for Zn(II) with metal ligands
contributed by each protein, and Zn(II) enhances IIAGlc inhibition [Feese, M.,
Pettigrew, D. W., Meadow, N. D., Roseman, S., and Remington, S. J. (1994) Proc.
Natl. Acad. Sci. U.S.A. 91, 3544-3548]. Here, we show that the Zn(II) enhancement
can be described quantitatively by a model with binding of Zn(II) to the complex 
with an apparent dissociation constant of less than 1 microM at pH 7.0 and 25
degreesC. Initial velocity studies show that IIAGlc is an uncompetitive inhibitor
with respect to both substrates, and the mechanism of inhibition is not altered
by Zn(II). The Zn(II)-liganding residue contributed by glycerol kinase (Glu478)
is substituted by using site-directed mutagenesis to construct the enzymes E478C,
E478D, E478H, and E478Q. The substitutions have only small effects on the
inhibition by IIAGlc in the absence of Zn(II), the catalytic properties, or other
allosteric regulation. However, all of the substitutions abolish the Zn(II)
enhancement of IIAGlc inhibition, and the X-ray crystallographic structures of
the complexes of IIAGlc with the E478C and E478H mutants show these substitutions
abolish binding of Zn(II) to the intermolecular site. These results support the
hypothesis that Zn(II) enhances the affinity for complex formation by binding at 
the intermolecular site, i.e., cation promoted association. The high affinity for
Zn(II) binding to the complex and the ability of the other four amino acid
residues to efficiently substitute for Glu478 in all functions except binding of 
Zn(II) suggest that cation promoted association of these two proteins may have a 
role in inducer exclusion in vivo.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 9538005 [PubMed - indexed for MEDLINE]

46: Anal Biochem. 1998 Apr 10;258(1):48-52.

Enzymatic synthesis of [3H]Cytidine 5'-diphospho-1, 2-diacyl-sn-glycerol.

Zhao M, Rua D, Hajra AK, Greenberg ML.

Department of Biological Sciences, Wayne State University, Detroit, Michigan
48202, USA.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol; CDP-DG) is an
important intermediate in the biosynthesis of the major glycerophosphate-based
phospholipids of prokaryotes and eukaryotes. This compound is expensive to
purchase and inefficient to prepare chemically. Radiolabeled CDP-diacylglycerol
is unavailable commercially. We describe a simple and inexpensive method to
synthesize [3H]CDP-DG enzymatically. The three-step enzymatic procedure includes 
phosphorylation of [3H]glycerol to sn-[3H]glycerol 3-phosphate (G3P) by
glycerokinase,acylation of [3H]G3P to [3H]phosphatidic acid (PA) by G3P
acyltransferase, and conversion of [3H]PA and CTP to [3H]CDP-DG by CDP-DG
synthase. This procedure is considerably less labor intensive and less expensive 
than is chemical synthesis, and the yield is at least 30%. Copyright 1998
Academic Press.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 9527846 [PubMed - indexed for MEDLINE]

47: Biochim Biophys Acta. 1998 Feb 17;1382(2):186-90.

Thermostable glycerol kinase from Thermus flavus: cloning, sequencing, and
expression of the enzyme gene.

Huang HS, Kabashima T, Ito K, Yin CH, Nishiya Y, Kawamura Y, Yoshimoto T.

School of Pharmaceutical Sciences, Nagasaki University, Japan.

The thermostable glycerol kinase (EC 2.7.1.30) gene from Thermus flavus was
cloned and expressed in Escherichia coli DH5 alpha. An open reading frame of 1488
bp for the glycerol kinase gene (glpK) starting with an ATG methionine codon was 
found, which encodes a protein of 496 amino acid residues whose calculated
molecular weight is 54,835. The amino acid sequence of T. flavus glycerol kinase 
is 80.6% and 64.1% identical with those of Bacillus subtilis and E. coli.
Transformants of E. coli DH5 alpha harboring plasmid pGYK12 with a 1505 bp
chromosomal DNA fragment containing the T. flavus glycerol kinase gene showed
about 23.8-fold higher glycerol kinase activity than T. flavus.

PMID: 9540790 [PubMed - indexed for MEDLINE]

48: Arch Biochem Biophys. 1998 Jan 15;349(2):236-45.

Conserved active site aspartates and domain-domain interactions in regulatory
properties of the sugar kinase superfamily.

Pettigrew DW, Smith GB, Thomas KP, Dodds DC.

Department of Biochemistry and Biophysics, Texas A&M University, College Station 
77843-2128, USA. DPETTIGREW@TAMU.EDU

The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes
show that the active site is located in a deep cleft between two domains whose
relative movement defines a domain closure conformational change thought to be
involved in the catalytic and regulatory properties of members of the
superfamily. To investigate the role of the domain closure in the regulatory
behavior, site-directed mutagenesis is used to alter specific domain-domain
interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol
3-phosphotransferase), a member of this superfamily. Two active site aspartate
residues are conserved throughout the superfamily, one (Asp245 in glycerol
kinase) which is proposed to act as a general base during catalysis and one
(Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound
Mg(II)-nucleotide complex. Each of these residues participates in domain-domain
interactions that are mediated by the bound substrates. The enzymes containing
the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by
affinity chromatography, and the effects of the substitutions on the catalytic
properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate 
(FBP), and the glucose-specific phosphocarrier protein, IIIGlc (also known as
IIAGlc), were determined. Each of the residues participates in catalysis;
kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the
D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone
shows that the level of activity seen for the D245N mutant enzyme is not due to
deamidation of the substituted asparagine. Each of the substitutions has little
effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N
substitution does not affect the extent of inhibition by IIIGlc. However, the
D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100
to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The
different sensitivities of the extents of FBP and IIIGlc inhibition to
perturbation of a domain-domain interaction mediated by Asp10 suggest that the
relations of the actions of these allosteric effectors to the domain closure
conformational change are different.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 9448710 [PubMed - indexed for MEDLINE]

49: Biochemistry. 1997 Dec 23;36(51):16087-96.

Structural studies of the Escherichia coli signal transducing protein IIAGlc:
implications for target recognition.

Feese MD, Comolli L, Meadow ND, Roseman S, Remington SJ.

Institute of Molecular Biology and Departments of Physics and Chemistry,
University of Oregon, Eugene, Oregon 97403, USA.

In Escherichia coli, the glucose-specific phosphocarrier protein of the
phosphotransferase system (PTS), IIAGlc (IIIGlc in older literature), is also the
central regulatory protein of the PTS. Depending upon its state of
phosphorylation, IIAGlc binds to a number of different proteins that display no
apparent sequence homology. Previous structural studies suggested that
nonspecific hydrophobic interactions, specific salt bridges, and an
intermolecular Zn(II) binding site contribute to the wide latitude in IIAGlc
binding sites. Two new crystal forms of IIAGlc have been solved at high
resolution, and the models were compared to those previously studied. The major
intermolecular contacts in the crystals differ in detail, but all involve the
hydrophobic active site of IIAGlc interacting with a hydrophobic patch on a
neighbor and all are shown to be surprisingly similar to the physiologically
relevant regulatory interaction of IIAGlc with glycerol kinase. In two crystal
forms, a helix on one molecule interacts with the face of another, while in the
other crystal form, the primary crystal contact consists of a strand of
beta-sheet that contributes to an intermolecular Zn(II) binding site with
tetrahedral ligation identical to that of the zinc peptidase thermolysin. Thus,
relatively nonspecific hydrophobic interactions combined with specific salt
bridges and an intermolecular cation binding site (cation-promoted association)
permit a regulatory protein to bind to target proteins that have little or no
sequence or structural homology with one another. It is suggested that signal
transduction by IIAGlc is a binary switch in which phosphorylation at the active 
site directly controls binding to target molecules.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 9405042 [PubMed - indexed for MEDLINE]

50: Biochemistry. 1997 Jun 10;36(23):6947-53.

Expression, purification, and characterization of enzyme IIA(glc) of the
phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum.

Zhu PP, Nosworthy N, Ginsburg A, Miyata M, Seok YJ, Peterkofsky A.

Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute,
National Institutes of Health, Bethesda, Maryland 20892, USA.

The gene encoding enzyme IIA(glc) (EIIA) of the phosphoenolpyruvate:sugar
phosphotransferase system of Mycoplasma capricolum was cloned into a regulated
expression vector. The purified protein product of the overexpressed gene was
characterized as an active phosphoacceptor from HPr with a higher pI than
previously described EIIAs. M. capricolum EIIA was unreactive with antibodies
directed against the corresponding proteins from either Gram-positive or
Gram-negative bacteria. Enzyme IIA(glc) behaved as a homogeneous, monomeric
species of 16,700 Mr in analytical ultracentrifugation. The circular dichroism
far-UV spectrum of EIIA reflects a low alpha-helical content and predominantly
beta-sheet structural content: temperature-induced changes in ellipticity at 205 
nm showed that the protein undergoes reversible, two-state thermal unfolding with
Tm = 70.0 +/- 0.3 degrees C and a van't Hoff deltaH of 90 kcal/mol. Enzyme I
(64,600 Mr) from M. capricolum exhibited a monomer-dimer-tetramer association at 
4 and 20 degrees C with dimerization constants of log K(A) = 5.6 and 5.1 [M(-1)],
respectively, in sedimentation equilibrium experiments. A new vector, capable of 
introducing an N-terminal His tag on a protein, was developed in order to
generate highly purified heat-stable protein (HPr). No significant interaction of
EIIA with HPr was detected by gel-filtration chromatography, intrinsic
tryptophanyl residue fluorescence changes, titration calorimetry, biomolecular
interaction, or sedimentation equilibrium studies. While Escherichia coli EIIA
inhibits Gram-negative glycerol kinase activity, the M. capricolum EIIA has no
effect on the homologous glycerol kinase. The probable regulator of sugar
transport systems, HPr(Ser) kinase, was demonstrated in extracts of M. capricolum
and Mycoplasma genitalium. Gene mapping studies demonstrated that, in contrast to
the clustered arrangement of genes encoding HPr and enzyme I in E. coli, these
genes are located diametrically opposite in the M. capricolum chromosome.

PMID: 9188690 [PubMed - indexed for MEDLINE]

51: J Biol Chem. 1997 May 30;272(22):14166-74.

Cloning and sequencing of two enterococcal glpK genes and regulation of the
encoded glycerol kinases by phosphoenolpyruvate-dependent, phosphotransferase
system-catalyzed phosphorylation of a single histidyl residue.

Charrier V, Buckley E, Parsonage D, Galinier A, Darbon E, Jaquinod M, Forest E,
Deutscher J, Claiborne A.

Institut de Biologie et Chimie des Protéines, CNRS, 7 passage du Vercors, F-69367
Lyon Cedex 07, France.

The glpK genes of Enterococcus casseliflavus and Enterococcus faecalis, encoding 
glycerol kinase, the key enzyme of glycerol uptake and metabolism in bacteria,
have been cloned and sequenced. The translated amino acid sequences exhibit
strong homology to the amino acid sequences of other bacterial glycerol kinases. 
After expression of the enterococcal glpK genes in Escherichia coli, both
glycerol kinases were purified and were found to be phosphorylated by enzyme I
and the histidine-containing protein of the phosphoenolpyruvate:glycose
phosphotransferase system. Phosphoenolpyruvate-dependent phosphorylation caused a
9-fold increase in enzyme activity. The site of phosphorylation in glycerol
kinase of E. casseliflavus was determined as His-232. Site-specific mutagenesis
was used to replace His-232 in glycerol kinase of E. casseliflavus with an
alanyl, glutamate, or arginyl residue. The mutant proteins could no longer be
phosphorylated confirming that His-232 of E. casseliflavus glycerol kinase
represents the site of phosphorylation. The His232 --> Arg glycerol kinase
exhibited an about 3-fold elevated activity compared with wild-type glycerol
kinase. Fructose 1,6-bisphosphate was found to inhibit E. casseliflavus glycerol 
kinase activity. However, neither EIIAGlc from E. coli nor the EIIAGlc domain of 
Bacillus subtilis had an inhibitory effect on glycerol kinase of E.
casseliflavus.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 9162046 [PubMed - indexed for MEDLINE]

52: Microbiology. 1997 Apr;143 ( Pt 4):1287-97.

Structure and gene-polypeptide relationships of the region encoding glycerol
diffusion facilitator (glpF) and glycerol kinase (glpK) of Pseudomonas
aeruginosa.

Schweizer HP, Jump R, Po C.

Department of Microbiology, Colorado State University, Fort Collins 80523, USA.
hschweizer@vines.colostate.edu

The glycerol facilitator is one of the few known examples of bacterial solute
transport proteins that catalyse facilitated diffusion across the cytoplasmic
membrane. A second protein, glycerol kinase, is involved in entry of external
glycerol into cellular metabolism by trapping glycerol in the cytoplasm as
sn-glycerol 3-phosphate. Evidence is presented that glycerol transport in
Pseudomonas aeruginosa is mediated by a similar transport system. The genes
encoding the glycerol facilitator, glpF, and glycerol kinase, glpK, were isolated
on a 4.5 kb EcoRI fragment from a chromosomal mini-library by functional
complementation of an Escherichia coli glpK mutant after establishing a map of
the chromosomal glpFK region with the help of a PCR-amplified glpK segment. The
nucleotide sequence revealed that glpF is the promoter-proximal gene of the glpFK
operon. The glycerol facilitator and glycerol kinase were identified in a T7
expression system as proteins with apparent molecular masses of 25 and 56 kDa,
respectively. The identities of the glycerol facilitator and glycerol kinase
amino acid sequences with their counterparts from Escherichia coli were 70 and
81%, respectively; this similarity extended to two homologues in the genome
sequence of Haemophilus influenzae. A chromosomal delta glpFK mutant was isolated
by gene replacement. This mutant no longer transported glycerol and could no
longer utilize it as sole carbon and energy source. Two ORFs, orfX and orfY,
encoding a putative regulatory protein and a carbohydrate kinase of unknown
function, were located upstream of the glpFK operon.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't

PMID: 9141691 [PubMed - indexed for MEDLINE]

53: J Biol Chem. 1996 Dec 27;271(52):33446-56.

Structures of active site histidine mutants of IIIGlc, a major signal-transducing
protein in Escherichia coli. Effects on the mechanisms of regulation and
phosphoryl transfer.

Pelton JG, Torchia DA, Remington SJ, Murphy KP, Meadow ND, Roseman S.

Bone Research Branch, National Institute of Dental Research, National Institutes 
of Health, Bethesda, Maryland 20892, USA.

IIIGlc (also called IIAGlc), a major signal-transducing protein in Escherichia
coli, is also a phosphorylcarrier in glucose uptake. The crystal and NMR
structures of IIIGlc show that His90, the phosphoryl acceptor, adjoins His75 in
the active site. Glutamine was substituted for His-, giving H75QIIIGlc and
H90QIIIGlc, respectively (Presper, K. A., Wong, C.-Y., Liu, L., Meadow, N. D.,
and Roseman, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4052-4055), but the
mutants showed unexpected properties. H90QIIIGlc loses regulatory functions of
IIIGlc, and the phosphoryltransfer rates between HPr/H75QIIIGlc are 200-fold less
than HPr/IIIGlc (Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271,
33440-33445). X-ray crystallography, differential scanning calorimetry, and NMR
have now been used to determine the structures of the mutants (phospho-H75QIIIGlc
was studied by NMR). The three methods gave completely consistent results. Except
for the His to Gln substitutions, the only significant structural changes were in
a few hydrogen bonds. H90QIIIGlc contains two structured water molecules (to
Gln90), which could explain its inability to regulate glycerol kinase.
Phospho-IIIGlc contains a chymotrypsin-like, hydrogen bond network
(Thr73-His75-O--phosphoryl), whereas phospho-H75QIIIGlc contains only one bond
(Gln75-O--phosphoryl). Hydrogen bonds play an essential role in a proposed
mechanism for the phosphoryltransfer reaction.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 8969208 [PubMed - indexed for MEDLINE]

54: J Bacteriol. 1996 May;178(10):2846-52.

A single amino acid change in Escherichia coli glycerol kinase abolishes glucose 
control of glycerol utilization in vivo.

Pettigrew DW, Liu WZ, Holmes C, Meadow ND, Roseman S.

Department of Biochemistry & Biophysics, Texas A&M University, College Station
77843-2128, USA. PETTIGREW@BIOCH.TAMU.EDU

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase)
is a key element in glucose control of glycerol metabolism. Its catalytic
activity is inhibited allosterically by the glycolytic intermediate, fructose
1,6-biphosphate, and by the phosphotransferase system phosphocarrier protein,
IIIGlc (also known as IIAGlc). These inhibitors provide mechanisms by which
glucose blocks glycerol utilization in vivo. We report here the cloning and
sequencing of the glpK22 gene isolated from E. C. C. Lin strain 43, a strain that
shows the loss of glucose control of glycerol utilization. DNA sequencing shows a
single missense mutation that translates to the amino acid change Gly-304 to Ser 
(G-304-S) in glycerol kinase. The effects of this substitution on the functional 
and physical properties of the purified mutant enzyme were determined. Neither of
the allosteric ligands inhibits it under conditions that produce strong
inhibition of the wild-type enzyme, which is sufficient to explain the phenotype 
of strain 43. However, IIIGlc activates the mutant enzyme, which could not be
predicted from the phenotype. In the wild-type enzyme, G-304 is located 1.3 nm
from the active site and 2.5 nm from the IIIGlc binding site (M. Feese, D. W.
Pettigrew, N. D. Meadow, S. Roseman, and S. J. Remington, Proc. Natl. Acad. Sci. 
USA 91:3544-3548, 1994). It is located in the same region as amino acid
substitutions in the related protein DnaK which alter its catalytic and
regulatory properties and which are postulated to interfere with a domain closure
motion (A. S. Kamath-Loeb, C. Z. Lu, W.-C. Suh, M. A. Lonetto, and C. A. Gross,
J. Biol. Chem. 270:30051-30059, 1995). The global effect of the G-304-S
substitution on the conformation and catalytic and regulatory properties of
glycerol kinase is consistent with a role for the domain closure motion in the
molecular mechanism for glucose control of glycerol utilization.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 8631672 [PubMed - indexed for MEDLINE]

55: Microbiology. 1996 Feb;142 ( Pt 2):217-30.

Catabolite repression and inducer control in Gram-positive bacteria.

Saier MH Jr, Chauvaux S, Cook GM, Deutscher J, Paulsen IT, Reizer J, Ye JJ.

Department of Biology, University of California at San Diego, La Jolla
92093-0116, USA. msaier@ucsd.edu

Results currently available clearly indicate that the metabolite-activated
protein kinase-mediated phosphorylation of Ser-46 in HPr plays a key role in
catabolite repression and the control of inducer levels in low-GC Gram-positive
bacteria. This protein kinase is not found in enteric bacteria such as E. coli
and Salmonella typhimurium where an entirely different PTS-mediated regulatory
mechanism is responsible for catabolite repression and inducer concentration
control. In Table 2 these two mechanistically dissimilar but functionally related
processes are compared (Saier et al., 1995b). In Gram-negative enteric bacteria, 
an external sugar is sensed by the sugar-recognition constituent of an Enzyme II 
complex of the PTS (IIC), and a dephosphorylating signal is transmitted via the
Enzyme IIB/HPr proteins to the central regulatory protein, IIAGlc. Targets
regulated include (1) permeases specific for lactose, maltose, melibiose and
raffinose, (2) catabolic enzymes such as glycerol kinase that generate
cytoplasmic inducers, and (3) the cAMP biosynthetic enzyme, adenylate cyclase
that mediates catabolite repression (Saier, 1989, 1993). In low-GC Gram-positive 
bacteria, cytoplasmic phosphorylated sugar metabolites are sensed by the HPr
kinase which is allostericlaly activated. HPr becomes phosphorylated on Ser-46,
and this phosphorylated derivative regulates the activities of its target
proteins. These targets include (1) the PTS, (2) non-PTS permeases (both of which
are inhibited) and (3) a cytoplasmic sugar-P phosphatase which is activated to
reduce cytoplasmic inducer levels. Other important targets of HPr(ser-P) action
are (4) the CcpA protein and probably (5) the CepB transcription factor. These
two proteins together are believed to determine the intensity of catabolite
repression. Their relative importance depends on physiological conditions. Both
proteins may respond to the cytoplasmic concentration of HPr(ser-P) and
appropriate metabolites. CepA possibly binds sugar metabolites such as FBP as
well as HPr(ser-P). Because HPr(his-P, ser-P) does not bind to CepA, the
regulatory cascade is also sensitive to the external PTS sugar concentration.
Mutational analyses (unpublished results) suggest that CepA may bind to a site
that includes His-15. Interestingly, both the CepA protein in the Gram-positive
bacterium, B. subtilis, and glycerol kinase in the Gram-negative bacterium, E.
coli, sense both a PTS protein and a cytoplasmic metabolic intermediate. The same
may be true of target permeases and enzymes in both types of organisms, but this 
possibility has not yet been tested. The parallels between the Gram-negative and 
Gram-positive bacterial regulatory systems are superficial at the mechanistic
level but fundamental at the functional level. Thus, the PTS participates in
regulation in both cases, and phosphorylation of its protein constituents plays
key roles. However, the stimuli sensed, the transmission mechanisms, the central 
PTS regulatory proteins that effect allosteric regulation, and some of the target
proteins are completely different. It seems clear that these two transmission
mechanisms evolved independently. They provide a prime example of functional
convergence.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.
    Review

PMID: 8932696 [PubMed - indexed for MEDLINE]

56: Prikl Biokhim Mikrobiol. 1996 Jan-Feb;32(1):110-5.

[Biochemical response of recombinant Hansenula polymorpha strains to
oversynthesis of homologous dioxyacetone kinase and bacterial beta-galactosidase]

[Article in Russian]

Vel'kov VV, Matys VIu, Sokolov DM.

Changes in the activities of key enzymes responsible for utilization of methanol 
by recombinant strains of methylotrophic yeasts H. polymorpha R22-2B and H.
polymorpha LAC-56 grown in a chemostat are described. The strain R22-2B
displaying a high activity of dioxyacetone kinase had also a high activity of
formaldehyde dehydrogenase, which increased the rate of dissimilation of
formaldehyde. There was a decrease in ATP concentration in the strain LAC-56
oversynthesizing beta-galactosidase from Escherichia coli; this effect decreased 
the rate of assimilation of formaldehyde.

Publication Types: 
    English Abstract

PMID: 8637840 [PubMed - indexed for MEDLINE]

57: Mol Gen Genet. 1995 Jul 28;248(2):236-41.

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of
the phosphotransferase system in Salmonella typhimurium LT2.

van der Vlag J, Postma PW.

E.C. Slater Institute, University of Amsterdam, The Netherlands.

In Enterobacteriaceae the nonphosphorylated form of IIAGlc of the
phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the
uptake and subsequent metabolism of glycerol and maltose by binding to, and
inhibiting, glycerol kinase and the MalK protein of the maltose transport system,
respectively. In this report we show that the IIAGlc-like domain of the
membrane-bound IIN-acetylglucosamine (IINag) of the PTS can replace IIAGlc in a
Salmonella typhimurium crr mutant strain that lacks all soluble IIAGlc. The
inhibition was most severe in cells which were partially induced for the glycerol
or maltose uptake systems. The Streptococcus thermophilus lactose transporter
LacS, which also contains a IIAGlc-like domain, could not replace IIAGlc. Neither
IINag nor LacS could replace IIAGlc in activation of adenylate cyclase.

PMID: 7651347 [PubMed - indexed for MEDLINE]

58: Microbiology. 1995 May;141 ( Pt 5):1193-8.

Mutations in the glycerol kinase gene restore the ability of a ptsGHI mutant of
Bacillus subtilis to grow on glycerol.

Wehtje C, Beijer L, Nilsson RP, Rutberg B.

Department of Microbiology, Lund University, Sweden.

Although glycerol is not taken up via the phosphotransferase system (PTS) in
Bacillus subtilis, some mutations that affect the general components of the PTS
impair the ability of cells to grow on glycerol. Five revertants of a pts
deletion mutant that grow on glycerol were analysed. They were shown to carry
mutations in the glycerol kinase gene. These are missense mutations located in
parts of the glpK gene that could encode regions important for the activity of
glycerol kinase. The results strongly suggest that the main effect of the PTS on 
glycerol utilization in B. subtilis is mediated via glycerol kinase.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't

PMID: 7773413 [PubMed - indexed for MEDLINE]

59: Dev Biol Stand. 1995;85:129-33.

Molecular analysis of enterococcal loci involved in novel catabolic pathways.

Claiborne A, Buckley E, Parsonage D, Ross RP, Ward DP.

Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem,
NC, USA.

Publication Types: 
    Review

PMID: 8586163 [PubMed - indexed for MEDLINE]

60: Biochemistry. 1994 Aug 23;33(33):10120-6.

Escherichia coli glycerol kinase: role of a tetramer interface in regulation by
fructose 1,6-bisphosphate and phosphotransferase system regulatory protein
IIIglc.

Liu WZ, Faber R, Feese M, Remington SJ, Pettigrew DW.

Department of Biochemistry & Biophysics, Texas A&M University, College Station
77843-2128.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase)
is a key element in a signal transduction pathway that couples expression of
genes required for glycerol metabolism to the relative availability of glycerol
and glucose. Its catalytic activity is inhibited by protein-protein interactions 
with IIIglc, a phosphotransferase system protein, and by fructose
1,6-bisphosphate (FBP); each of these allosteric effectors constitutes a positive
signal that glucose is available. Loss of glucose inhibition of glycerol
metabolism was used to screen for regulatory mutants of glycerol kinase after
hydroxylamine mutagenesis of the cloned glpK gene. Two mutant enzymes were
identified and shown by DNA sequencing to contain the mutations alanine 65 to
threonine (A65T) and aspartate 72 to asparagine (D72N). Initial velocity studies 
show the mutations do not significantly affect the catalytic properties, hence
active-site structures, of the enzymes. Both mutations decrease inhibition by
FBP; A65T eliminates the inhibition while D72N appears to decrease the affinity
for FBP and the extent of the inhibition. However, neither mutation significantly
affects inhibition by IIIglc. Gel-permeation chromatography studies show that
both of the mutations alter the dimer-tetramer assembly reaction of the enzyme
and the effect of FBP in increasing the molecular weight. The effects of the
mutations on the assembly reaction are consistent with the locations of these two
amino acid residues in the X-ray structure, which shows them to be associated
with an alpha-helix that constitutes one of the two subunit-subunit interfaces
within the tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 8060980 [PubMed - indexed for MEDLINE]

61: Genetics. 1994 Aug;137(4):903-17.

Microbial evolution in a simple unstructured environment: genetic differentiation
in Escherichia coli.

Rosenzweig RF, Sharp RR, Treves DS, Adams J.

Department of Biology, University of Michigan, Ann Arbor 48109.

Populations of Escherichia coli initiated with a single clone and maintained for 
long periods in glucose-limited continuous culture, become polymorphic. In one
population, three clones were isolated and by means of reconstruction experiments
were shown to be maintained in stable polymorphism, although they exhibited
substantial differences in maximum specific growth rates and in glucose uptake
kinetics. Analysis of these three clones revealed that their stable coexistence
could be explained by differential patterns of the secretion and uptake of two
alternative metabolites acetate and glycerol. Regulatory (constitutive and null) 
mutations in acetyl-coenzyme A synthetase accounted for different patterns of
acetate secretion and uptake seen. Altered patterns in glycerol uptake are most
likely explained by mutations which result in quantitative differences in the
induction of the glycerol regulon and/or structural changes in glycerol kinase
that reduce allosteric inhibition by effector molecules associated with
glycolysis. The evolution of resource partitioning, and consequent polymorphisms 
which arise may illustrate incipient processes of speciation in asexual
organisms.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 7982572 [PubMed - indexed for MEDLINE]

62: Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3544-8.

Cation-promoted association of a regulatory and target protein is controlled by
protein phosphorylation.

Feese M, Pettigrew DW, Meadow ND, Roseman S, Remington SJ.

Institute of Molecular Biology, University of Oregon, Eugene 97403.

A central question in molecular biology concerns the means by which a regulatory 
protein recognizes different targets. IIIGlc, the glucose-specific phosphocarrier
protein of the bacterial phosphotransferase system, is also the central
regulatory element of the PTS. Binding of unphosphorylated IIIGlc inhibits
several non-PTS proteins, but there is little or no sequence similarity between
IIIGlc binding sites on different target proteins. The crystal structure of
Escherichia coli IIIGlc bound to one of its regulatory targets, glycerol kinase, 
has been refined at 2.6-A resolution in the presence of products, adenosine
diphosphate and glycerol 3-phosphate. Structural and kinetic analyses show that
the complex of IIIGlc with glycerol kinase creates an intermolecular Zn(II)
binding site with ligation identical to that of the zinc peptidase thermolysin.
The zinc is coordinated by the two active-site histidines of IIIGlc, a glutamate 
of glycerol kinase, and a water molecule. Zn(II) at 0.01 and 0.1 mM decreases the
Ki of IIIGlc for glycerol kinase by factors of about 15 and 60, respectively. The
phosphorylation of one of the histidines of IIIGlc, in its alternative role as
phosphocarrier, provides an elegant means of controlling the cation-enhanced
protein-protein regulatory interaction. The need for the target protein to supply
only one metal ligand may account for the lack of sequence similarity among the
regulatory targets of IIIGlc.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 8170944 [PubMed - indexed for MEDLINE]

63: Nat Genet. 1993 Aug;4(4):367-72.

Genomic scanning for expressed sequences in Xp21 identifies the glycerol kinase
gene.

Guo W, Worley K, Adams V, Mason J, Sylvester-Jackson D, Zhang YH, Towbin JA, Fogt
DD, Madu S, Wheeler DA, et al.

Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas
77030.

Rapid genomic scanning methods are required to identify expressed sequences and
we report an efficient, sensitive and specific approach which relies upon
hybridization of an amplified, labeled cDNA library to digested cosmid DNA. We
identified expressed sequences within a cosmid in the glycerol kinase (GK)
"critical region" of Xp21 that had impressive similarity to prokaryotic GKs. We
used this genomic sequence information to clone the human hepatic GK cDNA.
Independent confirmation of the identity of this gene was obtained by functional 
complementation of GK deficient E. coli mutants with a construct containing the
complete human X-linked GK coding sequence.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 8401584 [PubMed - indexed for MEDLINE]

64: Curr Genet. 1993 Jul-Aug;24(1-2):21-5.

The glycerol kinase (GUT1) gene of Saccharomyces cerevisiae: cloning and
characterization.

Pavlik P, Simon M, Schuster T, Ruis H.

Institut für Biochemie und Molekulare Zellbiologie der Universität Wien, Austria.

The GUT1 gene of Saccharomyces cerevisiae, encoding glycerol kinase, was cloned
and sequenced. The cloned genomic DNA fragment contains an open reading frame
potentially coding for a protein of 709 amino acids with homology to bacterial
glycerol kinases (40.8% identity over 502 amino acids, and 42.1% identity over
496 amino acids, in comparison to the smaller E. coli and B. subtilis enzymes).
Disruption of GUT1 showed that the gene is required for growth on glycerol, but
not on glucose or ethanol media. No glycerol kinase activity was detected in the 
disruption mutant. According to enzyme activity and transcript analysis,
synthesis of glycerol kinase is repressed by glucose, and derepression is
ADR1-dependent.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 8358828 [PubMed - indexed for MEDLINE]

65: Hum Mol Genet. 1993 Feb;2(2):107-14.

Comment in:
    Hum Mol Genet. 1993 Feb;2(2):95-6.

Isolation of the human Xp21 glycerol kinase gene by positional cloning.

Walker AP, Muscatelli F, Monaco AP.

ICRF Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital,
Oxford, UK.

The gene for human glycerol kinase deficiency (GK) maps in Xp21.3 in a critical
region of about 50-250 kb located distal to the Duchenne muscular dystrophy gene 
(DMD) by analysis of patient deletions and YAC contigs. We have used a genomic
exon amplification strategy to isolate potential exons from two cosmids which
mapped to this interval. The genomic exons were used to isolate six overlapping
cDNA clones from human fetal liver which encode the X-linked glycerol kinase
gene. The cDNA clones map to cosmids, YAC clones and deletions in patients which 
define the GK critical region and also hybridize to several autosomal fragments
and one Xq fragment in genomic DNA. The GK gene is expressed most in human liver 
with three transcript sizes of 1.85, 2.7, and 3.7 kb. Sequence analysis of 1.5 kb
of several overlapping liver cDNA clones predicted a protein with approximately
63% similarity to the E. coli and B. subtilis glycerol kinase genes. The liver
cDNA clones have sequence identity with four genomic exons and the 3'
untranslated region from an Xp21.3 cosmid thus indicating that this is the
expressed GK gene which when deleted in patients gives rises to GK deficiency.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't

PMID: 8499898 [PubMed - indexed for MEDLINE]

66: J Bacteriol. 1993 Feb;175(4):1087-94.

Glycerol kinase of Escherichia coli is activated by interaction with the glycerol
facilitator.

Voegele RT, Sweet GD, Boos W.

Department of Biology, University of Konstanz, Germany.

Glycerol transport is commonly cited as the only example of facilitated diffusion
across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol
facilitator and glycerol kinase, are involved in the entry of external glycerol
into cellular metabolism. The glycerol facilitator is thought to act as a carrier
or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps
the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the
kinetics of glycerol uptake in a facilitator-minus strain are significantly
different from the kinetics of glycerol uptake in the wild type. Free glycerol
was not observed inside wild-type cells transporting glycerol, and diffusion of
glycerol across the cytoplasmic membrane was not the rate-limiting step for
phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol
phosphorylation are different, depending on the presence or absence of the
facilitator protein. We conclude that there is an interaction between the
glycerol facilitator protein and glycerol kinase that stimulates kinase activity,
analogous to the hexokinase- and glycerol kinase-porin interactions in
mitochondria.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't

PMID: 8432702 [PubMed - indexed for MEDLINE]

67: Science. 1993 Jan 29;259(5095):673-7.

Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol
kinase.

Hurley JH, Faber HR, Worthylake D, Meadow ND, Roseman S, Pettigrew DW, Remington 
SJ.

Institute of Molecular Biology, University of Oregon, Eugene 97403.

The phosphocarrier protein IIIGlc is an integral component of the bacterial
phosphotransferase (PTS) system. Unphosphorylated IIIGlc inhibits non-PTS
carbohydrate transport systems by binding to diverse target proteins. The crystal
structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in
complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was
determined. GK contains a region that is topologically identical to the adenosine
triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and
actin. IIIGlc binds far from the catalytic site of GK, indicating that long-range
conformational changes mediate the inhibition of GK by IIIGlc. GK and IIIGlc are 
bound by hydrophobic and electrostatic interactions, with only one hydrogen bond 
involving an uncharged group. The phosphorylation site of IIIGlc, His90, is
buried in a hydrophobic environment formed by the active site region of IIIGlc
and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding 
to GK by directly disrupting protein-protein interactions.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 8430315 [PubMed - indexed for MEDLINE]

68: J Bacteriol. 1992 Nov;174(21):6981-91.

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella
flexneri.

Truniger V, Boos W, Sweet G.

Department of Biology, University of Konstanz, Germany.

We have identified a new gene, glpX, belonging to the glp regulon of Escherichia 
coli, located directly downstream of the glpK gene. The transcription of glpX is 
inducible with glycerol and sn-glycerol-3-phosphate and is constitutive in a glpR
mutant. glpX is the third gene in the glpFKX operon. The function of GlpX remains
unknown. GlpX has an apparent molecular weight of 40,000 on sodium dodecyl
sulfate-polyacrylamide gels. In addition to determining the E. coli glpX
sequence, we also sequenced the corresponding glpFKX region originating from
Shigella flexneri, which after transfer into E. coli was instrumental in
elucidating the function of glpF in glycerol transport (D. P. Richey and E. C. C.
Lin, J. Bacteriol. 112:784-790, 1972). Sequencing of the glpFKX region of this
hybrid strain revealed an amber mutation instead of the tryptophan 215 codon in
glpF. The most striking difference between the E. coli and S. flexneri DNA was
found directly behind glpK, where two repetitive (REP) sequences were present in 
S. flexneri, but not in the E. coli sequence. The presence or absence of these
REP sequences had no effect on transport or on growth on glycerol. Not including 
the REP sequence-containing region, only 1.1% of a total of 2,167 bp sequenced
was different in the two sequences. Comparison of the sequence with those in the 
EMBL data library revealed a 99% identity between the last third of glpX and the 
first part of a gene called mvrA. We show that the cloned mvrA gene (M. Morimyo, 
J. Bacteriol. 170:2136-2142, 1988) originated from the 88-min region of the
Escherichia coli chromosome and not, as reported, from the 7-min region and that 
the gene product identified as MvrA is in fact encoded by a gene distal to glpX.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't

PMID: 1400248 [PubMed - indexed for MEDLINE]

69: J Biol Chem. 1992 Mar 25;267(9):6122-31.

Structure and regulation of the glpFK operon encoding glycerol diffusion
facilitator and glycerol kinase of Escherichia coli K-12.

Weissenborn DL, Wittekindt N, Larson TJ.

Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and
State University, Blacksburg 24061-0308.

The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 
with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein
which facilitates the diffusion of glycerol into the cell. The glpK gene encodes 
glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of
the operon, including the control region, the glpF gene, and part of the glpK
gene, was determined. The facilitator was predicted to contain 281 amino acids
with a calculated molecular weight of 29,780. It is a highly hydrophobic protein 
with a minimum of six potential transmembrane alpha helices. The transcription
start site for the glpFK operon was located 71 base pairs upstream from the
proposed translation start codon for glpF. Preceding the transcription start site
were sequences similar to the -10 and -35 consensus sequences for bacterial
promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the
glp repressor were identified by DNase I footprinting. The region protected by
the cAMP.CRP complex contained tandem sequences resembling the consensus sequence
for CRP binding. The CRP sites were centered at 37.5 and 60.5 base pairs upstream
of the start of transcription. The glp repressor protected an extensive area (-89
to -7 relative to the start point of transcription), sufficient for the binding
of four repressor tetramers. Two additional binding sites for the repressor were 
identified within the glpK coding region. The DNA containing these two operators 
synergistically increased the apparent affinity of glp repressor for DNA
fragments containing the four operators in the promoter region of the glpFK
operon. With this study, a total of 13 operators for the glp regulon have been
characterized. Comparison of these operators revealed the consensus
5'-WATGTTCGWT-3' for the operator half-site (W = A or T). The relative affinity
of the glp repressor for the various glp operators was assessed in vivo using a
promoter-probe vector. The relative apparent affinity of the control regions for 
glp repressor was glpFK greater than glpD greater than glpACB greater than glpTQ.
The degree of catabolite repression for each of the operons was assessed using a 
similar system. In this case, the relative sensitivity of the glp operons to
catabolite repression was glpTQ greater than glpFK greater than glpACB greater
than glpD.

Publication Types: 
    Comparative Study
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.

PMID: 1372899 [PubMed - indexed for MEDLINE]

70: Mol Microbiol. 1991 May;5(5):1081-9.

Analysis of the gluconate (gnt) operon of Bacillus subtilis.

Reizer A, Deutscher J, Saier MH Jr, Reizer J.

Department of Biology, University of California, San Diego, La Jolla 92093-0116.

The gluconate (gnt) operon of Bacillus subtilis includes the gntR, gntK, gntP,
and gntZ genes, respectively encoding the transcriptional repressor of the
operon, gluconate kinase, the gluconate permease, and an unidentified open
reading frame (Fujita and Fujita, 1987). We have compared the proteins encoded by
the gnt operon of B.subtilis with published sequences and showed that (i) the
gluconate repressor is homologous to several putative regulatory proteins in
Escherichia coli, (ii) the gluconate kinase of B. subtilis is homologous to
xylulose kinase, glycerol kinase and fucose kinase in E. coli (20-26% identity;
12-59 S.D.), (iii) the gluconate permease exhibits a C-terminal domain which is
homologous to a hydrophobic protein encoded by an unidentified open reading frame
(dsdAp) which precedes the dsdA gene of E. coli (39% identity; 19 S.D.), and (iv)
the gntZ gene product is homologous to 6-phosphogluconate dehydrogenases of other
bacteria and of animals (48-56%; 82-178 S.D.), thereby suggesting that the B.
subtilis gntZ encodes 6-phosphogluconate dehydrogenase. Several conserved regions
of the sequenced 6-phosphogluconate dehydrogenases can serve as signature
patterns of this protein. Computer analyses have indicated that the previously
reported sequences of the porcine and ovine 6-phosphogluconate dehydrogenases, as
well as the hypothetical DsdAp protein, are probably erroneous. The probable
reasons for the errors are reported along with the proposed revised sequences.

Publication Types: 
    Comparative Study
    Research Support, U.S. Gov't, P.H.S.

PMID: 1659648 [PubMed - indexed for MEDLINE]

71: Biochem J. 1991 Mar 15;274 ( Pt 3):819-24.

Compartmentalized system with membrane-bound glycerol kinase. Activity and
product distribution versus asymmetrical substrate supply.

Girard A, Merchie B, Maïsterrena B.

Laboratoire de Génie Enzymatique, Atelier de Biotechnologie, (CNRS-Université
Claude Bernard Lyon 1), Villeurbanne, France.

An artificial-membrane-bound glycerokinase chosen as a membrane-bound
two-substrate-enzyme model has been used to separate two unequal compartments of 
a specially designed diffusion cell. An interesting feature is the asymmetry of
compartments and the existence of a diffusion layer adjacent to only one face of 
the enzymic membrane. In such a situation the apparent enzyme activity and the
product distribution in the system have been studied versus all the possibilities
of combination of ATP and glycerol supply. Our approach has lead us to
differentiate two different roles played by a diffusion layer adjacent to a
permeable enzymic membrane. Depending on the spatial origin of the enzymic
substrates (i.e. from which compartment they derive), the diffusion layer can
play either the role of a passive additional resistance to that of the membrane
or the role of a third compartment in which the reaction product can partially
accumulate before splitting on both parts of the membrane. Our results mainly
demonstrate that a membrane-bound enzyme activity and the resulting product
distribution occurring in a compartmentalized system may be regulated by the
cumulative effect due to the asymmetry in volumes of the compartments, the
presence of a diffusion layer and the different possibilities of substrate
supply. With the topography studied, which is close to that reported for many 'in
vivo' situations, the product may be diffused lead to vectorial metabolism
processes.

PMID: 2012608 [PubMed - indexed for MEDLINE]

72: Biochemistry. 1990 Sep 18;29(37):8620-7.

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and
substrate binding studies.

Pettigrew DW, Yu GJ, Liu Y.

Department of Biochemistry and Biophysics, Texas A&M University, College Station 
77843-2128.

Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 
3-phosphotransferase) was investigated by using both kinetics and binding
methods. Initial-velocity studies in both reaction directions show a sequential
kinetic mechanism with apparent substrate activation by ATP and substrate
inhibition by ADP. In addition, the Michaelis constants differ greatly from the
substrate dissociation constants. Results of product inhibition studies and
dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme
has a random kinetic mechanism, which is consistent with the observed formation
of binary complexes with all the substrates and the glycerol-independent MgATPase
activity of the enzyme. Dissociation constants for substrate binding determined
by using ligand protection from inactivation by N-ethylmaleimide agree with those
estimated from the initial-velocity studies. Determinations of substrate binding 
stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP,
ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect
binding at a separate regulatory site. The random kinetic mechanism obviates the 
need to postulate such a site to explain the formation of binary complexes with
the nucleotides. The observed stoichiometry is consistent with a model for the
nucleotide regulatory behavior in which the dimer is the enzyme form present in
the assay and its subunits display different substrate binding affinities.
Several properties of the enzyme are consistent with negative cooperativity as
the basis for the difference in affinities. The possible physiological importance
of the regulatory behavior with respect to ATP is considered.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 2148683 [PubMed - indexed for MEDLINE]

73: J Bacteriol. 1990 Jan;172(1):424-30.

Glycerol facilitator of Escherichia coli: cloning of glpF and identification of
the glpF product.

Sweet G, Gandor C, Voegele R, Wittekindt N, Beuerle J, Truniger V, Lin EC, Boos
W.

Department of Biology, University of Konstanz, Federal Republic of Germany.

The glycerol facilitator is known as the only example of a transport protein that
catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here 
we show that the gene encoding the facilitator, glpF, is the first gene in an
operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. 
The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated
that it complemented a chromosomal glycerol transport-minus mutation, and
identified the gene product. The GlpF protein appeared in the membrane fraction
of plasmid-bearing strains and had an apparent Mr of 25,000.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 2152911 [PubMed - indexed for MEDLINE]

74: Biochemistry. 1989 Jul 11;28(14):5728-34.

Thiol and amino analogues as alternate substrates for glycerokinase from Candida 
mycoderma.

Knight WB, Cleland WW.

Institute for Enzyme Research, University of Wisconsin, Madison 53706.

The kinetic and catalytic mechanism of glycerokinase from Candida mycoderma was
examined with thiol and amino analogues of glycerol and with MgAMPPCP, an
analogue of MgATP. (S)-1-Aminopropanediol was phosphorylated on nitrogen (Vmax
0.4% that of glycerol) while the R enantiomer was phosphorylated on oxygen (Vmax 
0.7% that of glycerol). (S)-1-Mercaptopropanediol was phosphorylated on oxygen
(Vmax 3.5% that of glycerol), while the R enantiomer was phosphorylated on sulfur
(Vmax 0.001% that of glycerol). The hydroxyl group at C-2 thus orients the
substrate in the active site, while that at the carbon remote from
phosphorylation enhances both catalysis and binding of the substrate, presumably 
because of hydrogen-bonding interactions. The kinetic mechanism is random with a 
high degree of synergistic binding between the substrates, so that the mechanism 
appears ordered with glycerol adding first but equilibrium ordered with MgATP
binding first with the amino analogues.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 2550062 [PubMed - indexed for MEDLINE]

75: Nucleic Acids Res. 1989 Jun 12;17(11):4378.

Nucleotide sequence of the region encompassing the glpKF operon and its upstream 
region containing a bent DNA sequence of Escherichia coli.

Muramatsu S, Mizuno T.

Laboratory of Microbiology, School of Agriculture, Nagoya University, Japan.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 2544860 [PubMed - indexed for MEDLINE]

76: J Mol Biol. 1989 Jun 5;207(3):637-9.

Crystallization and preliminary X-ray studies of Escherichia coli glycerol
kinase.

Faber HR, Pettigrew DW, Remington SJ.

Department of Physics, University of Oregon, Eugene 97403.

Escherichia coli glycerol kinase, a major regulatory enzyme which catalyzes the
reversible MgATP-dependent phosphorylation of glycerol has been crystallized by
the hanging drop vapor diffusion method at room temperature. Three different
crystal forms have been obtained in the presence of glycerol and appear to be
suitable for X-ray crystallographic studies. Vapor diffusion against 55% ammonium
sulfate and 1% beta-octyl glucoside (pH 7.0) yields rhombohedral crystals with
space group R32, a = b = 277.1 A, c = 78.7 A (hexagonal indexing) containing a
dimer of Mr 112,000 in the asymmetric unit (Vm = 2.64 A3/dalton). Vapor diffusion
against sodium chloride in the presence of 10% (w/v) polyethylene glycol (pH 6.5 
to 7.0) yields two different crystal forms, both with space group P2(1). The
first form has a = 88.1 A, b = 99.3 A, c = 114.6 A, beta = 119 degrees, the
second form has a = 92.5 A, b = 117.6 A, c = 108.3 A, beta = 93.64 degrees.
Addition of ADP enhances growth of the monoclinic forms. These forms appear to
contain an entire tetramer of Mr 224,000 in the asymmetric unit and have Vm
values of 2.28 and 2.65 A3/dalton, respectively. All forms diffract to better
than 3.0 A resolution while the second monoclinic form diffracts to approximately
1.8 A.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.

PMID: 2547969 [PubMed - indexed for MEDLINE]

77: J Chromatogr. 1988 Jul 15;428(2):345-51.

Improved methods for purification and assay of glycerol kinase from Escherichia
coli.

Kee Y, Lee YS, Chung CH, Waxman L, Goldberg AL.

Department of Zoology, College of Natural Sciences, Seoul National University,
Korea.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 2851011 [PubMed - indexed for MEDLINE]

78: Eur J Biochem. 1988 Jun 1;174(2):387-9.

The stereochemical course of D-glyceraldehyde-induced ATPase activity of
glycerokinase from Escherichia coli.

Bethell RC, Lowe G.

Dyson Perrins Laboratory, University of Oxford, England.

D-Glyceraldehyde-induced hydrolysis of adenosine
(R)-5'-[gamma-17O,18O,thio]triphosphate catalysed by glycerokinase from
Escherichia coli gives inorganic [16O,17O,18O]thiophosphate with the
(S)-configuration, showing that the reaction proceeds with inversion of
configuration at phosphorus. This result provides powerful support for the
chemically most plausible mechanism, namely, that the hydrate of D-glyceraldehyde
is the effective substrate which after phosphorylation or thiophosphorylation
eliminates inorganic phosphate or inorganic thiophosphate, respectively, with
regeneration of D-glyceraldehyde.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 2838275 [PubMed - indexed for MEDLINE]

79: J Biol Chem. 1988 Jan 5;263(1):135-9.

Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the
primary structure of the enzyme.

Pettigrew DW, Ma DP, Conrad CA, Johnson JR.

Department of Biochemistry and Biophysics, Texas A&M University, College Station 
77843.

The glpK gene, which codes for Escherichia coli K-12 glycerol kinase (EC
2.1.7.30, ATP:glycerol 3-phosphotransferase), has been cloned into the HindIII
site of pBR322. The gene was contained in a 2.8-kilobase DNA fragment which was
obtained from a lambda transducing bacteriophage, lambda dglpK100 (Conrad, C.A., 
Stearns, G.W., III, Prater, W.E., Rheiner, J.A., and Johnson, J.R. (1984) Mol.
Gen. Genet. 195, 376-378). The DNA sequence of 2 kilobases of the cloned HindIII 
fragment was obtained using the dideoxynucleotide method. The start of the open
reading frame for the glpK gene was identified from the N-terminal sequence of
the first 22 amino acid residues of the purified enzyme, which was determined by 
automated Edman degradation. The open reading frame codes for a protein of 502
amino acids and a molecular weight of 56,106 which is in good agreement with the 
value previously determined by sedimentation equilibrium. The primary structure
of the protein as deduced from the gene sequence was corroborated by the
isolation and sequencing of four tryptic peptides, which were found to occur at
the following amino acid locations: 173-177, 203-211, 279-281, 464-468. The
N-terminal sequence of the purified enzyme shows that the enzyme undergoes
post-translational processing. Restriction digestion as well as DNA sequencing of
the supercoiled plasmid shows that the HindIII fragment is inserted into pBR322
such that the glpK gene is transcribed in a counterclockwise direction.
Examination of the upstream DNA sequence reveals two possible promoters of
essentially the same efficiency: the P1 promoter of pBR322 and a hybrid promoter 
which contains both bacterial and pBR322 DNA sequences.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 2826434 [PubMed - indexed for MEDLINE]

80: J Bacteriol. 1987 Jun;169(6):2488-93.

Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli.

Kalyananda MK, Engel R, Tropp BE.

When either 3H-labeled L-glyceraldehyde or 3H-labeled L-glyceraldehyde
3-phosphate (GAP) was added to cultures of Escherichia coli, the
phosphoglycerides were labeled. More than 81% of the label appeared in the
backbone of the phosphoglycerides. Chromatographic analyses of the labeled
phosphoglycerides revealed that the label was normally distributed into
phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results
suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is
converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the
bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an
NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was
specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as
a substrate. NADH could not replace NADPH as a coenzyme. The L-GAP:NADPH
oxidoreductase had an apparent Km of 28 and 35 microM for L-GAP and NADPH,
respectively. The enzyme was insensitive to sulfhydryl reagents and had a pH
optimum of approximately 6.6. The phosphonic acid analog of GAP,
3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an
apparent Km of 280 microM.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 3294792 [PubMed - indexed for MEDLINE]

81: Biochemistry. 1987 Mar 24;26(6):1723-7.

Inactivation of Escherichia coli glycerol kinase by
5'-[p-(fluorosulfonyl)benzoyl]adenosine: protection by the hydrolyzed reagent.

Pettigrew DW.

Incubation of Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol
3-phosphotransferase) with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo) at
pH 8.0 and 25 degrees C results in the loss of enzyme activity, which is not
restored by the addition of beta-mercaptoethanol or dithiothreitol. The FSO2BzAdo
concentration dependence of the inactivation kinetics is described by a mechanism
that includes the equilibrium binding of the reagent to the enzyme prior to a
first-order inactivation reaction in addition to effects of reagent hydrolysis.
The hydrolysis of the reagent has two effects on the observed kinetics. The first
effect is deviation from pseudo-first-order kinetic behavior due to depletion of 
the reagent. The second effect is the novel protection of the enzyme from
inactivation due to binding of the sulfonate hydrolysis product. The rate
constant for the hydrolysis reaction, determined independently from the kinetics 
of F- release, is 0.021 min-1 under these conditions. Determinations of the
reaction stoichiometry with 3H-labeled FSO2BzAdo show that the inactivation is
associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme
subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH,
5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO2BzAdo
provide protection from inactivation. The protection obtained with ATP is not
dependent on Mg2+. Less protection is obtained with glycerol, GMP, etheno-AMP,
and cAMP. No protection is obtained with CMP, UMP, TMP, etheno-CMP, GTP, or
fructose 1,6-bisphosphate. The results are consistent with modification by
FSO2BzAdo of a single adenine nucleotide binding site per enzyme subunit.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 3036208 [PubMed - indexed for MEDLINE]

82: Biochemistry. 1986 Aug 12;25(16):4711-8.

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic
acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites.

Pettigrew DW.

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia
coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by
N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The
inactivation by DTNB is reversed by dithiothreitol. In the cases of both
reagents, the kinetics of activity loss are pseudo first order. The dependencies 
of the rate constants on reagent concentration show that while the inactivation
by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the
enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate
constant shows a hyperbolic dependence on DTNB concentration. Complete
inactivation by each reagent apparently involves the modification of two
sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB
modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that
the inactivation is due to the modification of one sulfhydryl group per subunit, 
while two other groups are modified 6 and 15 times more slowly. The enzyme is
protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, 
AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol.
The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of
DTNB modification are different in the presence of glycerol or ATP, despite the
observation that the degree of protection afforded by both of these ligands is
the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 3021201 [PubMed - indexed for MEDLINE]

83: J Bacteriol. 1986 Jul;167(1):393-5.

Regulation of glycerol kinase by enzyme IIIGlc of the
phosphoenolpyruvate:carbohydrate phosphotransferase system.

de Boer M, Broekhuizen CP, Postma PW.

Wild-type glycerol kinase of Escherichia coli is inhibited by both
nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate
phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase,
resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to
inhibition by enzyme IIIGlc. The difference between the wild-type and mutant
enzymes was even greater when inhibition was measured in the presence of both
enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to
glycerol kinase required the presence of the substrate glycerol.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 3013838 [PubMed - indexed for MEDLINE]

84: Biochemistry. 1985 Jul 16;24(15):4148-55.

Formaldehyde metabolism by Escherichia coli. Carbon and solvent deuterium
incorporation into glycerol, 1,2-propanediol, and 1,3-propanediol.

Hunter BK, Nicholls KM, Sanders JK.

Escherichia coli were grown on 14.3% uniformly 13C-labeled glucose as the sole
carbon source and challenged anaerobically with 90% 13C-labeled formaldehyde. The
major multiply labeled metabolites were identified by 13C NMR spectroscopy to be 
glycerol and 1,2-propanediol, and a minor metabolite was shown to be
1,3-propanediol. In each case, formaldehyde is incorporated only into the C1
position. A novel form of 13C NMR isotope dilution analysis of the major products
reveals that all the 1,2-diol C1 is formaldehyde derived but that about 40% of
the glycerol C1 is derived from bacterial sources. Glycerokinase converted the
metabolite [1-13C]glycerol to equal amounts of [3-13C]glycerol 3-phosphate and
[1-13C]glycerol 3-phosphate, demonstrating that the metabolite is racemic. When
[13C]formaldehyde incubation was carried out in H2O/D2O mixtures, deuterium
incorporation was detected by beta- and gamma-isotope shifts. The 1,3-diol is
deuterium labeled only at C2 and only once, while the 1,2-diol and glycerol are
each labeled independently at both C2 and C3; C3 is multiply labeled. Deuterium
incorporation levels are different for each metabolite, indicating that the
biosynthetic pathways probably diverge early.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 3902080 [PubMed - indexed for MEDLINE]

85: Proc Natl Acad Sci U S A. 1985 Jul;82(13):4384-8.

ATP is essential for protein translocation into Escherichia coli membrane
vesicles.

Chen L, Tai PC.

The energy requirement for translocation of alkaline phosphatase and the outer
membrane protein OmpA into Escherichia coli membrane vesicles was studied under
conditions that permit posttranslational translocation and, hence, prior removal 
of various components necessary for protein synthesis. Translocation could be
supported by an ATP-generating system or, less well, by the protonmotive force
generated by D-lactate oxidation; the latter might act by generating ATP from
residual bound nucleotides. However, when protonmotive force inhibitors were used
or when ATP was further depleted by E. coli glycerol kinase, D-lactate no longer 
supported the translocation. Furthermore, ATP could still support protein
translocation in the presence of proton uncouplers or with membranes defective in
the F1 fraction of the H+-ATPase. We conclude that ATP is required for protein
translocation in this posttranslational system (and probably also in
cotranslational translocation); the protonmotive force may contribute but does
not appear to be essential.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 2861605 [PubMed - indexed for MEDLINE]

86: J Bacteriol. 1985 May;162(2):810-6.

Allosteric regulation of glycerol kinase by enzyme IIIglc of the
phosphotransferase system in Escherichia coli and Salmonella typhimurium.

Novotny MJ, Frederickson WL, Waygood EB, Saier MH Jr.

The mechanism by which enzyme IIIglc of the bacterial phosphotransferase system
regulates the activity of crystalline glycerol kinase from Escherichia coli has
been studied, and the inhibitory effects have been compared with those produced
by fructose-1,6-diphosphate. It was shown that the free, but not the
phosphorylated, form of enzyme IIIglc inhibits the kinase. Mutants of Salmonella 
typhimurium were isolated which were resistant to inhibition by either enzyme
IIIglc (glpKr mutants) or fructose-1,6-diphosphate (glpKi mutants), and each
mutant type was shown to retain full sensitivity to inhibition by the other
regulatory agent. Other mutants were fully or partially resistant to regulation
by both agents. The two regulatory sites on the kinase are evidently distinct but
must overlap or interact functionally. Kinetic analyses have revealed several
mechanistic features of the regulatory interactions. (i) Inhibition by both
allosteric regulatory agents is strongly pH dependent, with maximal inhibition
occurring at ca. pH 6.5 under the assay conditions employed. (ii) Binding of
enzyme IIIglc to glycerol kinase is also pH dependent, the Ki being near 4 microM
at pH 6.0 but near 10 microM at pH 7.0. (iii) Whereas fructose-1,6-diphosphate
inhibition apparently requires that the enzyme exist in a tetrameric state, both 
the dimer and the tetramer appear to be fully sensitive to enzyme IIIglc
inhibition. (iv) Inhibition by enzyme IIIglc (like that by
fructose-1,6-diphosphate) is noncompetitive with respect to both substrates. (v) 
The inhibitory responses of glycerol kinase to fructose-1, 6-diphosphate and
enzyme IIIglc show features characteristic of positive cooperativity at low
inhibitor concentration. (vi) Neither agent inhibits completely at high inhibitor
concentration. (vii) Apparent negative cooperativity with respect to ATP binding 
is observed with purified E. coli glycerol kinase, with glycerol kinase in crude 
extracts of wild-type S. typhimurium cells, and with glpKr and glpKi mutant forms
of glycerol kinase from S. typhimurium. These results serve to characterize the
regulatory interactions which control the activity of glycerol kinase by
fructose-1,6-diphosphate and by enzyme IIIglc of the phosphotransferase system.

Publication Types: 
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, P.H.S.

PMID: 2985549 [PubMed - indexed for MEDLINE]

87: J Bacteriol. 1984 Oct;160(1):55-60.

Purification and properties of dihydroxyacetone kinase from Klebsiella
pneumoniae.

Johnson EA, Burke SK, Forage RG, Lin EC.

Dihydroxyacetone (DHA) kinase of Klebsiella pneumoniae, a gene product of the dha
regulon responsible for fermentative dissimilation of glycerol and DHA, was
purified 120-fold to a final specific activity of 10 mumol X min-1 X mg of
protein-1 at 30 degrees C. The enzyme, a dimer of a 53,000 +/- 5,000-dalton
polypeptide, is highly specific for DHA (Km, ca.4 microM). Glycerol is not a
substrate at 1 mM and is not an inhibitor even at 100 mM. The enzyme is not
inhibited by 5 mM fructose-1,6-diphosphate. Ca2+ gives a higher enzyme activity
than Mg2+ as a cationic cofactor. Escherichia coli glycerol kinase acts on both
glycerol and DHA and is allosterically inhibited by fructose-1,6-diphosphate.
Antibodies raised against E. coli glycerol kinase cross-reacted with K.
pneumoniae glycerol kinase but not with K. pneumoniae DHA kinase.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 6090436 [PubMed - indexed for MEDLINE]

88: J Bacteriol. 1984 Apr;158(1):351-3.

Interaction between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase
system and glycerol kinase of Salmonella typhimurium.

Postma PW, Epstein W, Schuitema AR, Nelson SO.

Purified IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system of
Salmonella typhimurium inhibits glycerol kinase. Phosphorylation of IIIGlc via
phosphoenolpyruvate, enzyme I, and HPr abolishes this inhibition. The glycerol
facilitator is not inhibited by IIIGlc. It is proposed that regulation of
glycerol metabolism by the phosphoenolpyruvate:sugar phosphotransferase system is
at the level of glycerol kinase.

PMID: 6325396 [PubMed - indexed for MEDLINE]

89: J Gen Microbiol. 1984 Jan;130(1):83-8.

An inducible phosphoenolpyruvate: dihydroxyacetone phosphotransferase system in
Escherichia coli.

Jin RZ, Lin EC.

A phosphoenolpyruvate: dihydroxyacetone phosphotransferase was induced in
Escherichia coli grown on dihydroxyacetone as sole carbon source or in its
presence. This is the first example of a triose which can be acted upon by the
membrane complex to provide a central intermediate in glycolysis. The presence of
this system explains the ability of a mutant, in which the ATP-dependent glycerol
kinase is genetically replaced by a glycerol: NAD 2-oxidoreductase, to grow on
glycerol.

Publication Types: 
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 6368745 [PubMed - indexed for MEDLINE]

90: Mol Gen Genet. 1984;193(2):376-8.

Characterization of a glpK transducing phage.

Conrad CA, Stearns GW 3rd, Prater WE, Rheiner JA, Johnson JR.

A specialized glpK transducing phage, lambda glpK100, has been isolated and
characterized with respect to DNA structure. The glpK component of the glpKF
operon has been localized within a 2.0 kilobase pair (kbp) region of the
approximately 8.24 kbp bacterial DNA insert, and the positions of BamHI, EcoRI
and HindIII restriction sites within this DNA have been identified.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 6319974 [PubMed - indexed for MEDLINE]

91: J Bacteriol. 1982 Jun;150(3):1154-63.

Characteristics of a binding protein-dependent transport system for
sn-glycerol-3-phosphate in Escherichia coli that is part of the pho regulon.

Schweizer H, Argast M, Boos W.

The ugp-dependent transport system for sn-glycerol-3-phosphate has been
characterized. The system is induced under conditions of phosphate starvation and
in mutants that are constitutive for the pho regulon. The system does not operate
in membrane vesicles and is highly sensitive toward osmotic shock. The
participation of a periplasmic binding protein in the transport process can be
deduced from the isolation of transport mutants that lack the binding protein. As
with other binding protein-dependent transport systems, this protein appears to
be necessary but not sufficient for transport activity. The isolation of mutants 
has become possible by selection for resistance against the toxic analog
3,4-dihydroxybutyl-1-phosphonate that is transported by the system.
sn-Glycerol-3-phosphate transported via ugp cannot be used as the sole carbon
source. Strains have been constructed that lack alkaline phosphatase and glycerol
kinase. In addition, they are constitutive for the glp regulon and contain high
levels of glycerol-3-phosphate dehydrogenase. Despite the fact that these strains
exhibit high ugp-dependent transport activity for sn-glycerol-3-phosphate they
are unable to grow on it as a sole source of carbon. However, when cells are
grown on an alternate carbon source, (14)C label from
[(14)C]sn-glycerol-3-phosphate appears in phospholipids as well as in
trichloroacetic acid-precipitable material. The incorporation of (14)C label is
strongly reduced when sn-glycerol-3-phosphate is the only carbon source. In the
presence of an alternate carbon source, this inhibition is relieved, and
sn-glycerol-3-phosphate transported by ugp can be used as the sole source of
phosphate.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 7042685 [PubMed - indexed for MEDLINE]

92: Can J Microbiol. 1980 Mar;26(3):393-6.

Characterization of an Escherichia coli mutant which utilizes glycerol in the
absence of cyclic adenosine monophosphate.

Fraser AD, Yamazaki H.

The aerobic catabolism of glycerol depends on the expression of the glpK operon
specifying a glycerol kinase and the glpD operon specifying an
sn-glycerol-3-phosphate (G3P) dehydrogenase. It has not been clearly established 
how the expression of these operons is dependent on adenosine 3',5'-cyclic
monophosphate (cAMP). We have isolated a promoterlike mutant (CA8306B) which,
owing to a mutation in the glpK operon, can utilize glycerol in the absence of
cAMP. Glycerol kinase and G3P dehydrogenase are inducible in CA8306B and its
wild-type parent CA8000. The induced level of glycerol kinase in CA8306B is 30%
that of CA8000 and this level is increased fivefold by the addition of cAMP.
However, the induced level of G3P dehydrogenase in CA8306B is similar to that of 
CA8000 and is unaffected by cAMP addition. These results suggest that the
promotion of the glpK operon requires cAMP whereas the promotion of the glpD
operon does not.

Publication Types: 
    Research Support, Non-U.S. Gov't

PMID: 6250693 [PubMed - indexed for MEDLINE]

93: Biochemistry. 1980 Jan 22;19(2):325-9.

Stereochemical course of a phosphokinase using a chiral [18O]phosphorothioate.
Comparison with the transfer of a chiral [16O,17O,18O]phosphoryl group.

Pliura DH, Schomburg D, Richard JP, Frey PA, Knowles JR.

Synthetic adenosine 5'-O-[3-18O,3-thio]triphosphate having the R configuration at
the gamma-phosphorus has been used as a substrate in the reaction catalyzed by
glycerol kinase. The product sn-glycerol 3-[18O]phosphorothioate has been
isolated, and the configuration at phosphorus has been determined by ring closure
to the two diastereoisomeric cyclic 2,3-phosphorothioates of sn-glycerol and
analysis of the 18O content of each diastereoisomer. The structural identity of
these diastereoisomers has been determined by correlation with one of the
corresponding diastereoisomers of the cyclic 2,3-phosphorothioate of D-glycerate,
whose crystal structure is reported here. From these experiments it is evident
that glyc:rol kinase catalyzes the transfer of a thiophosphoryl group with
inversion of the configuration at phosphorus, in gratifying agreement with the
result from the transfer of a chiral [16O,17O,18O]phosphoryl group [Blättler, W. 
A., & Knowles, J. R. (1979) J. Am. Chem. Soc. 101, 510].

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 6243477 [PubMed - indexed for MEDLINE]

94: Biochemistry. 1979 Sep 4;18(18):3927-33.

Stereochemical course of phosphokinases. The use of adenosine
[gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the
reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate 
kinase.

Blättler WA, Knowles JR.

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an
isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group,
the configuration of which has been confirmed by independent stereochemical
analysis. This molecule has been used as a substrate in the reactions catalyzed
by glycerol kinase and by acetate kinase. The resulting samples of isotopically
labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as
substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl
groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been
determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) 
J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase
(and, by virtue of an earlier correlation, pyruvate kinase and hexokinase)
proceed by pathways that result in inversion of the configuration at phosphorus. 
The sterochemical approach provides an access to the otherwise cryptic events
that are involved in phosphoryl-group transfer within the ternary complexes of
these kinases and their substrates.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 226119 [PubMed - indexed for MEDLINE]

95: Biochemistry. 1978 Nov 28;17(24):5141-6.

Subunit dissociation in the allosteric regulation of glycerol kinase from
Escherichia coli. 2. Physical evidence.

de Riel JK, Paulus H.

The dependence of the molecular weight of glycerol kinase on enzyme concentration
and on binding of fructose 1,6-bisphosphate has been examined by velocity
sedimentation, gel filtration, and polyacrylamide gel electrophoresis. The
sedimentation coefficient and Stokes radius decrease as a consequence of dilution
in a manner consistent with dissociation into half-molecules, with limiting
values suggesting molecular weights of about 218,000 and 136,000 for the
associated and dissociated species, respectively. Fructose 1,6-bisphosphate (5
mM) prevents the decrease in sedimentation coefficient brought about by dilution,
suggesting a decrease in the apparent subunit dissociation constant of at least
four orders of magnitude. Electrophoretic mobility in polyacrylamide gels
increases as a consequence of dilution in the absence, but not in the presence,
of fructose 1,6-bisphosphate. Ferguson plots indicate that glycerol kinase has
the same molecular weight in the presence of fructose 1,6-bisphosphate as the
covalently cross-linked tetramer and is substantially smaller in the absence of
fructose 1,6-bisphosphate. These results are consistent with the model of
glycerol kinase proposed in the preceding paper of this issue [de Riel, J.K., and
Paulus, H. (1978), Biochemistry 17] relating subunit dissociation and ligand
binding.

Publication Types: 
    Research Support, U.S. Gov't, Non-P.H.S.

PMID: 215195 [PubMed - indexed for MEDLINE]

96: Biochemistry. 1978 Nov 28;17(24):5134-40.

Subunit dissociation in the allosteric regulation of glycerol kinase from
Escherichia coli. 1. Kinetic evidence.

de Riel JK, Paulus H.

Publication Types: 
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 215194 [PubMed - indexed for MEDLINE]

97: Biochemistry. 1978 Nov 28;17(24):5146-50.

Subunit dissociation in the allosteric regulation of Glycerol kinase from
Escherichia coli. 3. Role in desensitization.

de Riel JK, Paulus H.

The mechanism of desensitization of glycerol kinase to allosteric inhibition by
fructose 1,6-bisphosphate caused by salt, urea, and high pH has been examined in 
the light of the model proposed in an earlier paper [de Riel, J. K., and Paulus
H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding. KCl
(0.4 M) causes a tenfold decrease in the affinity of tetrameric glycerol kinase
for fructose, 1,6-bisphosphate but has no significant effect on the dissociation 
process itself. Urea (2 M) causes a large increase in the equilibrium constant
for the dissociation of the glycerol kinase tetramer to dimer but has no effect
on the affinity of the tetramer for the allosteric inhibitor. High pH (9--10) has
only a small effect on the subunit dissociation constant but greatly reduces the 
rates of subunit association and dissociation. Desensitization of glycerol kinase
to allosteric inhibition can thus occur by three different mechanisms, two of
which are directly related to the polysteric nature of the enzyme.

Publication Types: 
    Research Support, U.S. Gov't, Non-P.H.S.

PMID: 31903 [PubMed - indexed for MEDLINE]

98: J Bacteriol. 1978 Jul;135(1):239-50.

Cell-free synthesis of proteins related to sn-glycerol-3-phosphate transport in
Escherichia coli.

Schumacher G, Bussmann K.

An Escherichia coli periplasmic protein (GlpT) related to sn-glycerol-3-phosphate
transport was synthesized in a cell-free system directed by hybrid plasmic
ColE1-glpT DNA. The in vitro product cross-reacted with antisera against the
purified protein. The ColE1-glpT DNA-directed cell-free system was induced by
sn-glycerol-3-phosphate and phosphonomycin and was dependent on cyclic AMP. The
in vitro-synthesized protein showed the characteristics of a multimeric protein, 
as did the purified periplasmic protein. The main proportion of the newly
synthesized product had a higher molecular weight than the mature protein found
in the periplasm of cells and showed a more positive charge in two-dimensional
gel electrophoresis. Thus, a proportion of this protein is presumed to be
synthesized in vitro as a precursor. The cell-free system yielded a second
protein that is likely to be also coded for by the glpT operon. This protein had 
a molecular weight of approximately 33,000 in sodium dodecyl sulfate-acrylamide
gel electrophoresis and behaved like an intrinsic membrane protein.

PMID: 209011 [PubMed - indexed for MEDLINE]

99: J Bacteriol. 1977 Sep;131(3):1026-8.

Kinase replacement by a dehydrogenase for Escherichia coli glycerol utilization.

St Martin EJ, Freedberg WB, Lin EC.

A mutant of Escherichia coli that employs a glycerol:nicotinamide adenine
dinucleotide 2-oxidoreductase (EC 1.1.1.6), instead of adenosine
5'-triphosphate:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme 
for the dissimilation of glycerol was constructed. This mutant, like the
wild-type strain, still cannot grow anaerobically on glycerol without an
exogenous hydrogen acceptor.

Publication Types: 
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.

PMID: 197059 [PubMed - indexed for MEDLINE]

100: J Lipid Res. 1977 May;18(3):396-9.

A sensitive radioenzymatic assay for glycerol and acylglycerols.

Schneider PB.

A sensitive radioenzymatic assay for glycerol and acylglycerols is described. The
assay depends on the quantitative phosphorylation of glycerol to glycerophosphate
by glycerol kinase using [gamma-32P]ATP as a substrate. The 32P content of the
formed glycerophosphate is determined and gives a measure of the original
glycerol content. Acylglycerols can be determined by prior hydrolysis to
glycerol. The assay is sensitive to about 0.1 nmol of glycerol and can be
extended to 100 nmol. The assay can be applied to the determination of
acylglycerols separated by thin-layer chromatography in amounts as low as 0.5
nmol. The assay is particularly useful in the determination of the specific
activity of 14C- or 3H-labeled glycerol moeities.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 325162 [PubMed - indexed for MEDLINE]

101: Eur J Biochem. 1977 Feb;72(3):571-81.

Purification and properties of a periplasmic protein related to
sn-glycerol-3-phosphate transport in Escherichia coli.

Boos W, Hartig-Beecken I, Altendorf K.

Protein GLPT, a periplasmic protein previously recognized as closely related to
the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated 
by the cold osmotic shock procedure. It was purified by Sephadex chromatography
and isoelectric focussing. The purified protein does not exhibit any detectable
binding activity toward sn-glycerol-3-phosphate. It has no activity as a glycerol
phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the
presence of dodecylsulfate of the protein subsequent to treatment in urea,
boiling in dodecylsulfate and crosslinking indicates that it occurs as an
oligomeric protein composed of four identical subunits of 40 000 molecular
weight. Membrane vesicles of wild-type strains that contain protein GLPT in whole
cells loose it during vesicle preparation. However, they still exhibit high
transport activity toward sn-glycerol-3-phosphate. Membrane vesicles prepared
from glp T mutants that may or may not contain protein GLPT do not transport
sn-glycerol-3-phospahte. We conclude from these results that protein GLPT does
not participate in the energy-dependent active transport through the cytoplasmic 
membrane but could be involved in facilitating the diffusion of
sn-glycerol-3-phosphate through the outer layers of E. coli.

PMID: 190005 [PubMed - indexed for MEDLINE]

102: Eur J Biochem. 1976 May 17;65(1):207-12.

Metabolism of phosphatidylglycerol in cell-free extracts of Escherichia coli.

Luzon C, Ballesta JP.

Cell-free extracts from Escherichia coli strain number 9, lacking among other
enzymes glycerol kinase, are able to incorporate [2-3H] glycerol into
phospholipids. The characteristics of this incorporation indicate that it is not 
taking place through the regular glycerol phosphate pathway of phospholipid
synthesis which occurs when this compound is used as a precursor or even when
extracts of E. coli strain 7, having a functional glycerol kinase, are incubated 
with [2-3H] glycerol. In E. coli strain 9 extracts glycerol is exclusively
incorporated into the distal position of phosphatidyl-glycerol while in the other
strains the middle position glycerol is partially labelled.

PMID: 776627 [PubMed - indexed for MEDLINE]

103: Annu Rev Microbiol. 1976;30:535-78.

Glycerol dissimilation and its regulation in bacteria.

Lin EC.

Publication Types: 
    Research Support, U.S. Gov't, Non-P.H.S.
    Research Support, U.S. Gov't, P.H.S.
    Review

PMID: 825019 [PubMed - indexed for MEDLINE]

104: Eur J Biochem. 1975 Feb 21;51(2):449-57.

Glyceraldehyde phosphate at the reducing terminus of Salmonella Q haptens.
Salmonella montevideo.

Gmeiner J.

The O antigen polysaccharide of Salmonella montevideo was isolated from a
core-defective mutant by the phenol/water procedure, and was suspected to contain
phosphomonester and cyclic phosphodiester at its reducing end in anology to the O
hapten from Salmonella typhimurium (Kent and Obsborn, 1968. Therefore, it was
chromatographed on a DEAE-cellulose column. Whereas one part eluted with water
the other part of the polysaccharide could only be eluted with buffer. Both
fractions were further purified on Sephadex G100 and contained mannose, glucose, 
N-acetylglucosamine and phosphate in a molar ratio of 4:1:1: less than 0.1. In
order to specifically label the reducing end phosphate was removed enzymatically,
or the presumed cyclic diester was cleaved by mild hydrolysis, and the fractions 
were reduced with sodium horo[3H]hydride. Both fractions yield mainly
[3H]glycerol after hydrolysis and paper chromatogaphy. In addition, [3H]mannitol 
and [H]monohydroxyacetone could be identified by paper chromatography and were
concluded to be the result of phosphate migration and beta-elimination reactions 
taking place during the isolation procedure and the various treatments prior to
sodium boro[3H]hydride reduction. These findings in addition to periodate
oxidation studies indicated that the O antigen polysaccharide of Salmonella
montevideo had glyceraldehyde phosphate at its reducing end. From the
incorporation of 3H into the polysaccharide the O antigen was calculated to
consist of about 19 repeating units of 6 sugar residues each.

Publication Types: 
    Research Support, U.S. Gov't, P.H.S.

PMID: 1097244 [PubMed - indexed for MEDLINE]

105: Methods Enzymol. 1975;42:148-56.

Glycerol kinase.

Thorner JW.

PMID: 237175 [PubMed - indexed for MEDLINE]

106: Infect Immun. 1974 May;9(5):916-23.

Mutation in Shigella flexneri resulting in loss of ability to penetrate HeLa
cells and loss of glycerol kinase activity.

Kim R, Corwin LM.

A colonial variant of a virulent Shigella flexneri 2a has lost both virulence and
glycerol kinase activity. It also has several other altered characteristics:
lowered ability to oxidize tricarboxylic acid cycle intermediates, increased
electrophoretic mobility, and decreased sensitivity to sodium lauryl sulfate.
Genetic analysis has revealed that the gene governing glycerol kinase activity in
Shigella has a different chromosomal locus than that from Escherichia coli.
Furthermore, transduction of the Shigella glycerol kinase gene (glp K) into the
avirulent Shigella strain can restore the ability to penetrate HeLa cells,
whereas the gene from E. coli cannot. About half of the glp K mutants lose this
ability, and only about half of the revertants of an avirulent glp K mutant
regain it. This indicates that more than one gene affects glycerol kinase
activity in Shigella, only one of which is associated with penetration. Glycerol 
kinase activity is closely correlated with changes in electrophoretic mobility,
but does not appear to have any relationship to sodium lauryl sulfate sensitivity
nor to the oxidation of tricarboxylic acid cycle intermediates.

PMID: 4363235 [PubMed - indexed for MEDLINE]

107: J Bacteriol. 1974 Mar;117(3):1065-76.

Mutants of Escherichia coli defective in membrane phospholipid synthesis:
macromolecular synthesis in an sn-glycerol 3-phosphate acyltransferase Km mutant.

Bell RM.

sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected
from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted
from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective 
membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P 
was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane
preparations from the mutants than those of the parent. All extracts prepared
from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase
activity, but most contained less than 10% of the wild-type level. Membrane
preparations from revertants of the acyltransferase mutants had apparent K(m)'s
for G3P similar to that of the parent. Strains have been derived in which the G3P
requirement can be satisfied with glycerol in the presence of glucose, presumably
because the glycerol kinase was desensitized to inhibition by fructose
1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a
G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net
phospholipid synthesis stopped immediately; growth continued for about one
doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein
nearly doubled paralleling the growth curve; the rate of phospholipid synthesis
assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine
was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the
cells grew and then decreased as the cells stopped growing; the rate of protein
synthesis showed no increase and declined more slowly than the rates of RNA and
DNA synthesis when the cells stopped growing. The cells retained and gained in
the capacity to synthesize phospholipids upon glycerol deprivation. These data
indicate that net phospholipid synthesis is not required for continued
macromolecular synthesis for about one doubling, and that the rates of these
processes are not coupled during this time period.

PMID: 4591941 [PubMed - indexed for MEDLINE]

108: J Bacteriol. 1973 Sep;115(3):816-23.

Three kinds of controls affecting the expression of the glp regulon in
Escherichia coli.

Freedberg WB, Lin EC.

Three kinds of control mechanisms govern the expression of the members of the glp
regulon for glycerol and sn-glycerol 3-phosphate (G3P) catabolism in Escherichia 
coli K-12: specific repression by the product of the glpR gene; catabolite
repression; and respiratory repression (the effect exerted by exogenous hydrogen 
acceptors). The operons of the glp system show different patterns of response to 
each control. By growing in parallel a mutant strain with temperature-sensitive
repressor (glpR(ts)) and an isogenic control with a deletion in the regulator
gene at progressively higher temperatures, it was possible to show that the
synthesis of aerobic G3P dehydrogenase (glpD product) is far more sensitive to
specific repression than that of either glycerol kinase (glpK product) or G3P
transport (glpT product). Conversely, in the strain with a deletion in the
regulator gene, the syntheses of glycerol kinase and G3P transport are more
sensitive to catabolite repression than that of the aerobic G3P dehydrogenase.
The levels of the two flavoprotein G3P dehydrogenases vary in opposite directions
in response to changes of exogenous hydrogen acceptors. For example, the ratio of
the aerobic enzyme to the anaerobic enzyme (specified by glpA) is high when
molecular oxygen or nitrate serves as the hydrogen acceptor and low when fumarate
plays this role. This trend is not influenced by the addition of cyclic adenosine
3',5'-monophosphate to the growth medium. Thus, respiratory repression most
likely involves a third mechanism of control, independent of specific or
catabolite repression.

PMID: 4580569 [PubMed - indexed for MEDLINE]

109: J Biol Chem. 1973 Jun 10;248(11):3922-32.

Catalytic and allosteric properties of glycerol kinase from Escherichia coli.

Thorner JW, Paulus H.

Publication Types: 
    Comparative Study

PMID: 4575199 [PubMed - indexed for MEDLINE]

110: J Bacteriol. 1972 Nov;112(2):784-90.

Importance of facilitated diffusion for effective utilization of glycerol by
Escherichia coli.

Richey DP, Lin EC.

Wild-type Escherichia coli possesses an inducible permeation system which
catalyzes facilitated diffusion of glycerol into the cell. A spectrophotometric
method can be used to assess the presence of this mechanism. The structural gene 
for the facilitator (glpF) and the structural gene for glycerol kinase (glpK)
apparently belong to a single operon. The glpF(+) allele permits effective
glycerol utilization by the cells, and, at millimolar concentrations of glycerol,
cells carrying the glpF(+) allele grow much faster than glpF genotypes. Although 
the glycerol-scavenging power of the cell depends both on the facilitated entry
of the substrate and its subsequent trapping by an adenosine
triphosphate-dependent phosphorylation, the two gene products, the facilitator
and kinase, function independently. Wild-type Shigella flexneri appears to be
glpK(+) but glpF. This organism grows slowly in media at low concentrations of
glycerol. When the glpF(+) and glpK(+) alleles of E. coli are inserted into the
S. flexneri genome by transduction, the hybrid strain grows rapidly in low
glycerol medium. Vice versa, when the glpF and glpK(+) alleles of S. flexneri are
incorporated into E. coli, the hybrid strain grows slowly in low glycerol medium.

PMID: 4563976 [PubMed - indexed for MEDLINE]

111: Proc Natl Acad Sci U S A. 1972 Mar;69(3):648-51.

Mechanism of the enzymatic synthesis of cardiolipin in Escherichia coli.

Hirschberg CB, Kennedy EP.

In previous studies, the enzymatic conversion of phosphatidylglycerol to
cardiolipin (diphosphatidylglycerol) in cell-free preparations from E. coli was
shown to be stimulated by the addition of CDP-dipalmitin, suggesting the
participation of the cytidine coenzyme as phosphatidyl donor. The present
communication, however, presents three lines of evidence supporting the following
mechanism for the synthesis of cardiolipin in E. coli.2 Phosphatidylglycerol --> 
cardiolipin + glycerolWhen CDP-dipalmitin labeled with (32)P in the phosphatidyl 
moiety was incubated with phosphatidyl[2-(3)H]-glycerol, the cardiolipin produced
in the enzymatic reaction was labeled with tritium, but not with (32)P. Thus,
CDP-diglyceride stimulates the reaction but does not participate as phosphatidyl 
donor. When [(28)P]phosphatidyl[2-(3)H]glycerol was used as substrate, the ratio 
of tritium to (32)P in the cardiolipin product was only half of that in the
starting phosphatidylglycerol, consistent with the elimination of 1 mol of
glycerol during conversion to cardiolipin. Finally, free glycerol produced during
the reaction has been unambiguously identified by phosphorylation with ATP in a
reaction catalyzed by glycerol kinase (EC 2.7.1.30), followed by chromatographic 
isolation of labeled sn-3-glycero-3-phosphate.

PMID: 4551982 [PubMed - indexed for MEDLINE]

112: J Bacteriol. 1971 Dec;108(3):1338-47.

Properties and mode of action of a bactericidal compound (=methylglyoxal)
produced by a mutant of Escherichia coli.

Krymkiewicz N, Diéguez E, Rekarte UD, Zwaig N.

A lethal product (BPG) produced by a glycerol kinase mutant of Escherichia coli
was purified, and its mode of action on E. coli was studied. At concentrations
where BPG strongly inhibits in vivo deoxyribonucleic acid, ribonucleic acid, and 
protein synthesis, it produces small effects on other functions: slight
inhibition of respiration and small changes in intracellular pools of substrates,
nucleic acids degradation, and adenosine triphosphate levels. BPG also inhibits
in vitro protein synthesis and produces inactivation of bacteriophage T4. The
bactericidal product has been identified in another laboratory as methylglyoxal
(MG). By comparing BPG and MG, we confirmed this observation and concluded that
the activity found in our BPG preparation is due to its MG content. We also
observed that MG is able to react with guanosine triphosphate. According to these
results, it is interpreted that MG could act directly on macromolecular synthesis
by reacting with the guanine residues of nucleic acids and its precursors.

PMID: 4945198 [PubMed - indexed for MEDLINE]

113: J Bacteriol. 1971 Oct;108(1):137-44.

Lethal synthesis of methylglyoxal by Escherichia coli during unregulated glycerol
metabolism.

Freedberg WB, Kistler WS, Lin EC.

In Escherichia coli K-12, the conversion of glycerol to triose phosphate is
regulated by two types of control mechanism: the rate of synthesis of glycerol
kinase and the feedback inhibition of its activity by fructose-1,6-diphosphate. A
strain which has lost both control mechanisms by successive mutations, resulting 
in the constitutive synthesis of a glycerol kinase no longer sensitive to
feedback inhibition, can produce a bactericidal factor from glycerol. This toxic 
factor has been identified by chemical and enzymological tests as methylglyoxal. 
Methylglyoxal can be derived from dihydroxyacetone phosphate through the action
of an enzyme which is present at high constitutive levels in the extracts of the 
mutant as well as that of the wild-type strain. Nine spontaneous mutants
resistant to 1 mm exogenous methylglyoxal have been isolated. In all cases the
resistance is associated with increased levels of a glutathione-dependent
enzymatic activity for the removal of methylglyoxal. Methylglyoxal-resistant
mutants derived from the glycerol-sensitive parental strain also became immune to
glycerol.

PMID: 4941552 [PubMed - indexed for MEDLINE]

114: J Biol Chem. 1971 Jun 25;246(12):3885-94.

Composition and subunit structure of glycerol kinase from Escherichia coli.

Thorner JW, Paulus H.

PMID: 4934840 [PubMed - indexed for MEDLINE]

115: J Bacteriol. 1971 Jun;106(3):724-31.

Promoter-like mutant with increased expression of the glycerol kinase operon of
Escherichia coli.

Berman-Kurtz M, Lin EC, Richey DP.

A glycerol-specific phenotypic revertant isolated from a mutant of Escherichia
coli missing enzyme I of the phosphoenolpyruvate phosphotransferase system was
studied. This revertant is capable of producing higher levels of glycerol kinase 
and the protein mediating the facilitated diffusion of glycerol (facilitator)
than wild-type cells. The kinase of the revertant is indistinguishable from the
wild-type enzyme with respect to its sensitivity to feedback inhibition by
fructose-1,6-diphosphate, its pH optimum, and its turnover number. The synthesis 
of glycerol kinase in strains bearing the suppressor locus is resistant to
catabolite repression. The suppressor mutation mapped at the known glpK locus.
Thus, it is suggested that the mutation occurred in the promoter of the operon
specifying the kinase and the facilitator.

PMID: 4934061 [PubMed - indexed for MEDLINE]

116: J Bacteriol. 1970 Dec;104(3):1230-1235.

Role of Pyridine Nucleotides in the Control of Respiration in
Ultraviolet-Irradiated Escherichia coli B/r Cells.

Swenson PA, Schenley RL.

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830.

Escherichia coli B/r cells grown on glycerol-containing medium and irradiated
with ultraviolet light to about 1% survival respire for about 1 hr and then cease
completely for several hours. The results of studies on cell-free respiration and
analyses of pyridine nucleotide levels at various times after ultraviolet
irradiation show that the cessation of respiration is associated with two
changes-loss of glycerol kinase activity and complete disappearance of pyridine
nucleotides. Under other cultural conditions in which respiratory inhibition is
less complete and more transitory, the losses of pyridine nucleotides are smaller
and the rises which follow are correlated with increases in respiratory activity.

PMID: 16559097 [PubMed - as supplied by publisher]

117: J Bacteriol. 1970 Jun;102(3):753-9.

Glycerol kinase, the pacemaker for the dissimilation of glycerol in Escherichia
coli.

Zwaig N, Kistler WS, Lin EC.

The activity of glycerol kinase is rate-limiting in the metabolism of glycerol by
cells of Escherichia coli. A mutant strain producing a glycerol kinase resistant 
to inhibition by fructose-1,6-diphosphate grows faster than its wild-type parent 
on glycerol as the sole source of carbon and energy. The amount of intracellular 
fructose-1,6-diphosphate was determined for wild-type cells growing exponentially
on glycerol. The water content of such cells was also determined, allowing
calculation of the intracellular concentration of fructose-1,6-diphosphate. This 
value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type
glycerol kinase. The desensitization of glycerol kinase to feedback inhibition
also enhances the power of glycerol to exert catabolite repression, both on the
enzymes of the glycerol system itself and on those of the lactose system.
However, desensitization of glycerol kinase alone does not eliminate the
phenomenon of diauxic growth in a glucose-glycerol medium. Biphasic growth in
such a medium is abolished if the altered enzyme is produced constitutively. The 
constitutive production of the mutant kinase at high levels, however, renders the
cells vulnerable to glycerol. Thus, when the cells have been grown on a carbon
source with a low power for catabolite repression, e.g., succinate, sudden
exposure to glycerol leads to overconsumption of the nutrient and cell death.

PMID: 4914079 [PubMed - indexed for MEDLINE]

118: J Biol Chem. 1967 Mar 10;242(5):1030-5.

Purification and properties of glycerol kinase from Escherichia coli.

Hayashi SI, Lin EC.

PMID: 5335908 [PubMed - indexed for MEDLINE]

119: Science. 1966 Aug 12;153(737):755-7.

Feedback inhibition of glycerol kinase, a catabolic enzyme in Escherichia coli.

Zwaig N, Lin EC.

Fructose-1 ,6-diphosphate is a feedback inhibitor of the catabolic enzyme,
glycerol kinase, in Escherichia coli. A mutant was isolated which produced a
desensitized enzyme. Glucose was no longer as effective in preventing the
utilization of exogenous glycerol by cells which synthesized constitutively such 
an altered enzyme, even though the usual degree of catabolite repression still
operated.

Publication Types: 
    In Vitro

PMID: 5328677 [PubMed - indexed for MEDLINE]

120: J Bacteriol. 1966 Aug;92(2):470-476.

Properties of a Mutant of Escherichia coli with a Temperature-sensitive
Fructose-1,6-Diphosphate Aldolase.

Böck A, Neidhardt FC.

Department of Biological Sciences, Purdue University, Lafayette, Indiana.

Böck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt.
Properties of a mutant of Escherichia coli with a temperature-sensitive
fructose-1,6-diphosphate aldolase. J. Bacteriol. 92:470-476. 1966.-A mutant of
Escherichia coli in which fructose-1,6-diphosphate aldolase functions at 30 C but
not at 40 C was used to study the physiological effect of a specific block in the
Embden-Meyerhof glycolytic pathway. Growth of the mutant at 40 C was found to be 
inhibited by the presence of glucose or certain related compounds in the medium. 
At 40 C, glucose was metabolized at 30 to 40% of the control rate and was
abnormal in that glucose was converted into other six-carbon substances (probably
gluconate, in large part) that were released into the culture medium. The
inhibition was complete, but transient; its duration depended upon the initial
amount of inhibitor added. The resumption of growth at 40 C was correlated with
the further catabolism of the excreted compounds. When glycerol was used to grow 
the mutant at 40 C, the growth inhibition by glucose was accompanied by cessation
of glycerol metabolism. Growth on alpha-glycerol phosphate was not inhibited
under these conditions, implicating glycerol kinase as a possible site of
inhibition; no inhibition of glycerol kinase by sugar phosphates, however, could 
be detected in vitro. The inhibitory effect of glucose on growth at 40 C is not
caused by a deficit of intracellular adenosine triphosphate, but may be the
result of a generalized poisoning of many cell processes by a greatly increased
intracellular concentration of fructose-1,6-diphosphate, the substrate of the
damaged enzyme.

PMID: 16562137 [PubMed - as supplied by publisher]

121: J Bacteriol. 1966 May;91(5):1763-6.

Chromosomal location of the structural gene for glycerol kinase in Escherichia
coli.

Cozzarelli NR, Lin EC.

Cozzarelli, N. R. (Harvard Medical School, Boston, Mass.), and E. C. C. Lin.
Chromosomal location of the structural gene for glycerol kinase in Escherichia
coli. J. Bacteriol. 91:1763-1766. 1966.-A glycerol kinase mutant site has been
mapped by transduction and sexual conjugation. Three-factor crosses with the two 
procedures yielded the following gene order: arginine-1-methionine-1-glycerol
kinase-isoleucine, valine-16. An additional 13 independent glycerol kinase mutant
sites mapped in the same region. Since some of the mutants were able to produce a
protein serologically indistinguishable from the wild-type enzyme, it is
concluded that the region mapped represents the structural gene for the kinase.

Publication Types: 
    In Vitro

PMID: 5327904 [PubMed - indexed for MEDLINE]

122: J Mol Biol. 1965 Dec;14(2):515-21.

Product induction of glycerol kinase in Escherichia coli.

Hayashi SI, Lin EC.

Publication Types: 
    In Vitro

PMID: 4956458 [PubMed - indexed for MEDLINE]

123: J Bacteriol. 1965 Nov;90(5):1325-9.

Growth stasis by accumulated L-alpha-glycerophosphate in Escherichia coli.

Cozzarelli NR, Koch JP, Hayashi S, Lin EC.

Cozzarelli, N. R. (Harvard Medical School, Boston, Mass.), J. P. Koch, S.
Hayashi, and E. C. C. Lin. Growth stasis by accumulated l-alpha-glycerophosphate 
in Escherichia coli. J. Bacteriol. 90:1325-1329.1965.-Cells of Escherichia coli
K-12 can grow on either glycerol or l-alpha-glycerophosphate as the sole source
of carbon and energy. The first step in the dissimilation of glycerol requires a 
kinase, and the initial process of utilization of l-alpha-glycerophosphate
involves an active transport system. In either case, intracellular
l-alpha-glycerophosphate is an intermediate whose further metabolism depends upon
a dehydrogenase. When this enzyme is lost by mutation, the cells not only fail to
grow on glycerol or l-alpha-glycerophosphate, but are subject to growth
inhibition in the presence of either compound. Resistance to inhibition by
glycerol can be achieved by the loss of glycerol kinase. Such cells are still
susceptible to growth inhibition by l-alpha-glycerophosphate. Similarly, in
dehydrogenase-deficient cells, immunity to exogenous l-alpha-glycerophosphate can
be achieved by genetic blocking of the active transport system. Such cells are
still sensitive to free glycerol in the growth medium. Reversal of inhibition by 
glycerol or l-alpha-glycerophosphate in cells lacking the dehydrogenase can also 
be brought about by the addition of glucose. Glucose achieves this effect without
recourse to catabolite repression. Our results suggest that growth stasis
associated with the over-accumulation of l-alpha-glycerophosphate is due to
interference with other cellular processes by competition with physiological
substrates rather than to depletion of cellular stores of adenosine triphosphate 
or inorganic phosphate.

Publication Types: 
    In Vitro

PMID: 5321485 [PubMed - indexed for MEDLINE]