Glutamine amidotransferase activity of NAD+ synthetase from Mycobacterium tuberculosis depends on an amino-terminal nitrilase domain.
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.
Bellinzoni
Marco
M
Dipartimento di Genetica e Microbiologia, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy. mbellin@pasteur.fr
Buroni
Silvia
S
Pasca
Maria Rosalia
MR
Guglierame
Paola
P
Arcesi
Fabio
F
De Rossi
Edda
E
Riccardi
Giovanna
G
D019732:Amide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics
D005973:Glutamine; D000619:Aminohydrolases; C021867:nitrilase; D019731:Carbon-Nitrogen Ligases; D019732:Amide Synthases; C038479:NAD+ synthase; C020828:GMP synthase (glutamine-hydrolyzing)