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.