1. Res Microbiol. 2005 Mar;156(2):173-7.

Glutamine amidotransferase activity of NAD+ synthetase from Mycobacterium
tuberculosis depends on an amino-terminal nitrilase domain.

Bellinzoni M, Buroni S, Pasca MR, Guglierame P, Arcesi F, De Rossi E, Riccardi G.

Dipartimento di Genetica e Microbiologia, University of Pavia, Via Ferrata 1,
27100 Pavia, Italy. mbellin@pasteur.fr

NAD(+) synthetase (NadE; E.C. 6.3.5.1) from Mycobacterium tuberculosis utilizes
both glutamine and ammonia to catalyze NAD(+) production, in contrast to the
corresponding NH(3)-dependent enzymes from other prokaryotes. Here we report the 
site-directed mutagenesis of amino acids located in the N-terminal domain and
predicted to be essential for glutamine hydrolysis. The residues forming the
putative catalytic triad (Cys176, Glu52 and Lys121) were replaced by alanine; the
mutated enzymes were expressed in the Escherichia coli Origami (DE3) strain and
purified. The three mutants completely lost their glutamine-dependent activity,
clearly indicating that Cys176, Glu52 and Lys121 are crucial for this activity.
In contrast, the C176A and E52A variants, respectively, retained 90 and 30% of
the original NH(3)-dependent specific activity, while the K121A mutant lost this 
activity. The results show that glutamine-amidotransferase activity is mediated
by an N-terminal domain belonging to the superfamily of nitrilases. This domain, 
a new type of glutamine amide transfer (GAT) domain, is the first to be
characterized in bacterial NAD(+) synthetases.

PMID: 15748981 [PubMed - indexed for MEDLINE]