Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammonia-dependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammonia-dependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism. NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammonia-dependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammonia-dependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism. Molecular identification of human glutamine- and am monia-dependent NAD synthetases. Carbon-nitrogen hydrolase domain confers glutamine dependency.Journal of Biological ChemistryVol. 278Issue 42PreviewVol. 278 (2003) 10914–10921 Full-Text PDF Open Access nicotinic acid mononucleotide carbon-nitrogen hydrolase high performance liquid chromatography nicotinic acid adenine dinucleotide kilobase(s) The coenzyme NAD has a role in the majority of metabolic redox reactions and represents an essential component of metabolic pathways in all living cells. In a number of signaling pathways, NAD also serves as a precursor of potent calcium-mobilizing agents such as cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (1Lee H.C. Physiol. Rev. 1997; 77: 1133-1164Google Scholar) and serves as a substrate for post-translational modifications of protein, mono- (2Hara N. Tsuchiya M. Shimoyama M. J. Biol. Chem. 1996; 271: 29552-29555Google Scholar, 3Okazaki I.J. Moss J. J. Biol. Chem. 1998; 273: 23617-23620Google Scholar, 4Haag F. Koch-Nolte F. J. Biol. Regul. Homeost. Agents. 1998; 12: 53-62Google Scholar) and poly(ADP-ribosyl)ations (5Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Google Scholar). Depletion of cellular NAD by poly(ADP-ribosyl)transferase activation in response to DNA damage results in cell death (6Pieper A.A. Verma A. Zhang J. Snyder S.H. Trends Pharmacol. Sci. 1999; 20: 171-181Google Scholar). Increased NAD synthesis has been shown to extend life span in yeast (7Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Google Scholar) and in Caenorhabditis elegans(8Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Google Scholar) via activation of an NAD-dependent histone deacetylase, silent information regulator 2 (Sir2) (9Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Google Scholar). The cellular level of NAD may modulate the sensitivity of cells to apoptotic responses through deacetylation of the p53 tumor suppressor by a human homologue of Sir2 (10Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Google Scholar). Recent publications have demonstrated that fluctuation of the NAD level in cells seems to have significant impact on their physiology. Despite these significant effects of NAD levels on cellular functions, mechanisms regulating cellular contents of NAD through metabolic events remain to be established. NAD biosynthesis is accomplished through either de novo or salvage pathways (11Foster J.W. Moat A.G. Microbiol. Rev. 1980; 44: 83-105Google Scholar, 12White H.B. Everse J. Anderson B.M. You K.S. Pyridine Nucleotide Coenzyme: Biosynthesis of Salvage Pathways of Pyridine Nucleotide Coenzymes. Academic Press, Inc., New York1982: 1-17Google Scholar). These two pathways converge at the level of an intermediate nicotinic acid mononucleotide (NaMN),1 which is then converted into nicotinic acid adenine dinucleotide (NaAD) through the action of NaMN adenylyltransferase and, lastly, into NAD by NAD synthetase (Fig. 1). Although most of the genes involved in both pathways have been identified in procaryotes (13Tritz G.J. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1987: 557-563Google Scholar), little is known of those genes, including that of NAD synthetase in eucaryotes, except for nicotinamide mononucleotide adenylyltransferase (14Emanuelli M. Carnevali F. Saccucci F. Pierella F. Amici A. Raffaelli N. Magni G. J. Biol. Chem. 2001; 276: 406-412Google Scholar) and quinolinic acid phosphoribosyltransferase (15Fukuoka S. Nyaruhucha C.M. Shibata K. Biochim. Biophys. Acta. 1998; 1395: 192-201Google Scholar) genes. NAD synthetase catalyzes the conversion of NaAD into NAD, and NH3 or glutamine is used as an amide donor in the following reactions.NaAD+NH3+ATP→NAD+AMP+PPiREACTION1NaAD+glutamine+ATP→NAD+glutamate+AMP+PPiREACTION2In procaryotes, two types of NAD synthetase have been reported, a type catalyzes Reaction 1 and is strictly ammonia-dependent, whereas the other synthetase catalyzes both Reactions 1 and 2 and uses both ammonia and glutamine as amide donors. Bacillus subtilis synthetase, a representative of the former, is a protein of 271 amino acid residues (16Nessi C. Albertini A.M. Speranza M.L. Galizzi A. J. Biol. Chem. 1995; 270: 6181-6185Google Scholar), and crystal structure analysis of the synthetase revealed residues responsible for binding sites for ATP and NaAD (17Rizzi M. Nessi C. Mattevi A. Coda A. Bolognesi M. Galizzi A. EMBO J. 1996; 15: 5125-5134Google Scholar). Although one of the latter enzymes, Mycobacterium tuberculosis synthetase of 738 amino acids, has been also reported (18Cantoni R. Branzoni M. Labò M. Rizzi M. Riccardi G. J. Bacteriol. 1998; 180: 3218-3221Google Scholar), the structural basis underlying the potential to use glutamine or ammonia of two types of synthetase has not been determined. In eucaryotes, although NAD synthetase activity of the latter type has been reported (19Yu C.K. Dietrich L.S. J. Biol. Chem. 1972; 247: 4794-4802Google Scholar, 20Zerez C.R. Wong M.D. Tanaka K.R. Blood. 1990; 75: 1576-1582Google Scholar), molecular cloning and characterization have not been done. In the case of the strictly ammonia-dependent NAD synthetase, the counterpart ofB. subtilis enzyme, even the presence of the activity has not been demonstrated in eucaryotic organisms. We now report molecular identification of two human NAD synthetases, a synthetase that can use not only ammonia but also glutamine and the other synthetase with strictly ammonia-dependent activity (referred as NADsyn1 and NADsyn2, respectively). To our knowledge, this is the first report demonstrating the presence of the strictly ammonia-dependent NAD synthetase in eucaryotes. We also describe the structural basis underlying the potential of NADsyn1 to use glutamine as an amide donor as well as the distinct distribution of NADsyn1 and NADsyn2 in animal tissues. α-32PdCTP (6000 Ci/mmol) was purchased from Amersham Biosciences. NaAD, AMP and inorganic pyrophosphatase were from Sigma. ATP, NAD, l-glutamine, and ammonium chloride were from Oriental Yeast (Tokyo, Japan), Roche Molecular Biochemicals (Basel, Switzerland), Nacalai Tesque (Kyoto, Japan), and Wako Pure Chemical Industries (Osaka, Japan), respectively. COS-7 cells and a human promyelocytic leukemia cell line HL60 were obtained from Riken Cell Bank (Tsukuba Science City, Japan). Human glioma cell line LN229 and human hepatocyte cell lines HepG2 and Huh7 were from American Type Culture Collection (Manassas, VA). To express NADsyn1 and NADsyn2 as C-terminal-His6-tagged proteins in COS-7 cells, a His6 tag sequence followed by a TGA termination codon was introduced into the pcDNA3 vector (Invitrogen) between XbaI and ApaI cloning sites to obtain the of human NADsyn1 were from human two of and and and The were and to cloning and NADsyn1 Human NADsyn2 was from LN229 with by and cDNAs were and XbaI for NADsyn1 and and XbaI for NADsyn2 and and and The vector used to express the mutant in which Cys-175 was replaced with was a the vector NADsyn1 and the and the codon is indicated in The expression a were into COS-7 cells an to the the COS-7 cells were with and by the cells were by and NAD synthetases were from the with to the of glutamine-dependent NAD synthetase was from with by on the sequence of homologue of human NADsyn1 and of expression or were by in both was from from HL60 cells with The of the NADsyn1 and NADsyn2 cDNAs were with 2 and and the were into and were and NAD synthetase activity of the protein was on of the NAD as the mutant and NADsyn2 were with glutamine or as indicated in the 2 ATP, 1 NaAD, and of the of NADsyn1 activity, 2 was in the glutamine was used as a substrate for was of The reactions were by of and then at for to obtain the H. Scholar). The was for and for by The of NAD at known was used to the of NAD. NAD synthetase activity was by the NAD of from the NAD of the In NAD synthetase activity was by the NAD synthetase reactions been by a with the NaAD, and were on a Nacalai with acid as the and by at Kinetic for NAD synthetase were as by analysis of a of the of NAD synthesis. In the at of two of the of the substrate from to 2 for NaAD, from to 1 ATP, from 1 to for and from to and or from to for In the by for NaAD and ATP were glutamine as an amide donor, whereas in reactions by NADsyn2 and were of NAD were by the NAD synthetase reactions were by of on for and then by the were with or of pyrophosphatase at for The reactions were by and the inorganic phosphate was as by and Dietrich (19Yu C.K. Dietrich L.S. J. Biol. Chem. 1972; 247: 4794-4802Google Scholar). NADsyn1 and NADsyn2 were on and in in the presence of were into then in the either glutamine for NADsyn1 or 1 for NADsyn2 at for 2 NAD in the was the as from tissues and human cell lines were on a to and The was at for in and The were then at for in the to amino acids of glutamine-dependent NAD synthetase in or the of human NADsyn2 by with α-32PdCTP and an The were in 2 at for then with at for with the glutamine-dependent NAD synthetase or at for with NADsyn2 and to at with an these NADsyn2 not the and were a the amino acid sequence of B. subtilis NAD synthetase (16Nessi C. Albertini A.M. Speranza M.L. Galizzi A. J. Biol. Chem. 1995; 270: 6181-6185Google Scholar) number as a we found two human NAD synthetase, and in a corresponding to the and of the we as and obtained two human and NADsyn2, proteins of and amino acids, respectively. To the of the we of the cDNAs of In the of we corresponding to the used for of the In of NADsyn1 we we not codon but found that the codon was in a sequence M. J. Cell Biol. Scholar), and the codon was the first NADsyn2, we obtained an codon of the Thus, we that NADsyn1 and NADsyn2 of the In a of known functional and in protein structures, we identified the domain number and an ATP binding a sequence of a family of including NAD synthetase M.L. V.J. J.L. Biol. 1996; Scholar), in both NADsyn2 and the of NADsyn1 and sequence that most of the residues ATP binding and and NaAD binding sites and in B. subtilis synthetase (16Nessi C. Albertini A.M. Speranza M.L. Galizzi A. J. Biol. Chem. 1995; 270: 6181-6185Google Scholar) strictly in both NADsyn1 and NADsyn2 (Fig. 2 These that cDNAs for NADsyn1 and NADsyn2 NAD synthetases. We the domain and the as the synthetase In to the synthetase NADsyn1 only the carbon-nitrogen hydrolase domain number at the (Fig. 2 The with a cysteine residue essential for nitrilase activity M. N. T. H. M. H. T. H. Proc. Natl. Acad. Sci. U. S. A. Scholar), is shared with enzymes to the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia (Fig. 2 Sci. Scholar, C. R. A. 1995; Scholar). B. subtilis synthetase, which the domain ammonia but not glutamine (16Nessi C. Albertini A.M. Speranza M.L. Galizzi A. J. Biol. Chem. 1995; 270: 6181-6185Google Scholar), the presence of the domain in NADsyn1 but not in NADsyn2 suggested that the uses both glutamine and ammonia in NAD synthesis, whereas the latter is strictly the cysteine residue in the domain was also in NADsyn1 at a of the cysteine in NADsyn1 was to be essential for the use of glutamine. these sequence we (i) that both NADsyn1 and have NAD synthetase activity, (ii) that NADsyn1 uses not only ammonia but also whereas NADsyn2 uses only and (iii) that the of Cys-175 in NADsyn1 glutamine-dependent NAD synthetase activity with the ammonia-dependent activity We expressed NADsyn2, and a mutant NADsyn1 in which Cys-175 was replaced with in COS-7 cells as and we these proteins on analysis indicated that the and mutant NADsyn1 not have a molecular of in with the from the The NADsyn2 as a with a molecular of (Fig. the from the The may on the of the protein To NADsyn2, and have the we the proteins with glutamine or in the presence of NaAD and ATP then the NAD synthetase of the proteins a shown in the NADsyn1 the activity with either glutamine or the other the NADsyn2 NAD synthesis with (Fig. In with the the activity of the mutant NADsyn1 was not glutamine was used as a whereas the activity with (Fig. Kinetic analysis indicated that NADsyn1 a for glutamine for with NADsyn2 a for but a for glutamine NADsyn1 and NADsyn2 the for ATP and NaAD, in the of those reported for synthetases (16Nessi C. Albertini A.M. Speranza M.L. Galizzi A. J. Biol. Chem. 1995; 270: 6181-6185Google Scholar, C.K. Dietrich L.S. J. Biol. Chem. 1972; 247: 4794-4802Google Scholar). Kinetic of the mutant NADsyn1 obtained with not from those of the which that of the glutamine was not of a in the structure of with NADsyn2 the mutant synthetase a for the glutamine which to ammonia in the of ammonia in the in be to Thus, these NADsyn2 but not the mutant synthetase NAD ammonia (Fig. as was seen with subtilis synthetase (16Nessi C. Albertini A.M. Speranza M.L. Galizzi A. J. Biol. Chem. 1995; 270: 6181-6185Google in NAD synthetase reactions by the and mutant NADsyn1 and synthetase activity was the of and the was not synthetase activity was the of and the was not were as the of NAD synthetase activity was the of and the was not determined. Open in a The were as the of these results with our (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 not only ammonia but also glutamine as an amide donor, whereas NADsyn2 is an ammonia-dependent NAD synthetase, and (iii) that Cys-175 in NADsyn1 is essential for the to use glutamine as an amide In with our sequence we conclude that the domain in the of in is responsible for of glutamine as an amide donor and, confers glutamine on the synthetase, whereas the synthetase domain in NADsyn1 and NADsyn2 in NAD synthesis from ammonia. To the role of the domain in we a of the of NADsyn1 and NADsyn2 as well as a the or of of their we not these not We the of the reactions by the synthetases. The NADsyn1 and NADsyn2 were with glutamine and in the presence of NaAD and ATP and the were by shown in 1 of AMP and was 1 of NAD the by These results indicate that of NaAD by the enzymes is with ATP to AMP and as for the J. J. Biol. Chem. Scholar). of either ATP, NaAD, or amide donors from the in a of NAD synthesis by not To of human NADsyn1 and NADsyn2 as synthetases (16Nessi C. Albertini A.M. Speranza M.L. Galizzi A. J. Biol. Chem. 1995; 270: 6181-6185Google Scholar, C.K. Dietrich L.S. J. Biol. Chem. 1972; 247: 4794-4802Google Scholar, 20Zerez C.R. Wong M.D. Tanaka K.R. Blood. 1990; 75: 1576-1582Google Scholar), we the synthetases by and NAD synthetase activity in shown in of NADsyn1 and NADsyn2 with proteins of and that NADsyn1 and NADsyn2 may as a and a respectively. To the tissue distribution of NADsyn1 and NADsyn2, were with from tissues and human cell shown in a of was for with The sites of NADsyn1 expression were the small intestine, kidney, liver, and whereas the skeletal and a In the and small intestine, an additional was observed at The NADsyn1 was also expressed in human glioma and promyelocytic leukemia cell lines not Although an not a human with a high of analysis that an of is expressed in human cells and HepG2 and Huh7 cell (Fig. In the mouse, all of the tissues expressed the a homologue of In the and in skeletal the was observed of the not In and and human cell lines an additional was The amino acid of NADsyn1 and NADsyn2 of and amino acids, with those in and and 2 in a protein revealed that NADsyn1 significant amino acid to eucaryotic proteins from the and C. (Fig. of the NADsyn1 sequence with these proteins that the including ATP and NaAD binding sites in the and the essential cysteine residues for use of glutamine in the The domain of NADsyn1 also sequence to NAD synthetases from M. tuberculosis (18Cantoni R. Branzoni M. Labò M. Rizzi M. Riccardi G. J. Bacteriol. 1998; 180: 3218-3221Google Scholar) J.C. G. J. Bacteriol. Scholar) (Fig. 2 and in the cysteine residues were strictly in the two synthetases. NADsyn2 not significant sequence to other eucaryotic proteins the The present is the first identification of two NAD synthetases, NADsyn1 and NADsyn2, in expression of the synthetases indicated that although NADsyn1 both glutamine and ammonia as amide ammonia may not serve as a physiological amide donor for for In contrast, NADsyn2 uses ammonia more does NADsyn1 for but to be to use glutamine as a physiological amide donor glutamine Thus, we conclude that NADsyn1 is a glutamine-dependent NAD synthetase, whereas NADsyn2 is a strictly ammonia-dependent To our knowledge, this is the first for the presence of an ammonia-dependent NAD synthetase in eucaryotes. by the catalytic activity of NADsyn2, which with the mutant in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases M. N. T. H. Scholar, M. H. T. H. Proc. Natl. Acad. Sci. U. S. A. Scholar) was replaced with we identified the domain as the functional domain of NAD synthetase to from the amide of glutamine and, to use glutamine as an amide of the domain as the of glutamine that the of human NADsyn1 found in different also glutamine-dependent NAD synthetases. The domain in the NAD synthetase from M. known to NAD synthesis with glutamine (18Cantoni R. Branzoni M. Labò M. Rizzi M. Riccardi G. J. Bacteriol. 1998; 180: 3218-3221Google Scholar), now the structural basis underlying the glutamine of the These results that the glutamine-dependent NAD synthetases can be classified as a possible glutamine amidase into the nitrilase family. In the an cysteine residue has been to as a in the catalytic a is to a by in the of the M. N. T. H. Scholar, M. H. T. H. Proc. Natl. Acad. Sci. U. S. A. Scholar). the cysteine residue in the nitrilases is in these glutamine-dependent NAD synthetases, the cysteine residues in the synthetases a on a of ammonia from glutamine. In the synthetase of these synthetases, of NaAD in the presence of ATP, the ammonia in the domain the NaAD, in NAD, as J. J. Biol. Chem. Scholar). analysis and a analysis of NADsyn1 a of the catalytic of these glutamine-dependent NAD synthetases. We that NADsyn1 catalytic activity in a multimeric form. NAD synthetases from human and glutamine-dependent and, to also multimeric enzymes (19Yu C.K. Dietrich L.S. J. Biol. Chem. 1972; 247: 4794-4802Google Scholar, 20Zerez C.R. Wong M.D. Tanaka K.R. Blood. 1990; 75: 1576-1582Google Scholar). has been reported that nitrilase family a by through in the domain Sci. Scholar) (Fig. 2 Thus, that the including in of yeast NAD synthetase has been reported to have two an ammonia-dependent NAD synthetase and an additional and the latter has been to use glutamine as amide donor (19Yu C.K. Dietrich L.S. J. Biol. Chem. 1972; 247: 4794-4802Google Scholar, H. J.L. Biol. 1998; Scholar). the yeast homologue of human NADsyn1 with a molecular of has the seems that the represents the yeast synthetase, and is that the is required for glutamine-dependent NAD synthetase The of NADsyn1 expression revealed by analysis may in NAD animal tissues. expression of NADsyn1 was observed in the small intestine, liver, kidney, and whereas skeletal muscle and the very nicotinamide mononucleotide the of the substrate of NAD synthetase NaAD (Fig. has been reported to be expressed in skeletal muscle and the (14Emanuelli M. Carnevali F. Saccucci F. Pierella F. Amici A. Raffaelli N. Magni G. J. Biol. Chem. 2001; 276: 406-412Google Scholar), with NADsyn1 the of NAD synthesis in these tissues. NADsyn2 is expressed in skeletal muscle and in the heart. In these expression of the of ammonia from glutamine has also been demonstrated M. Physiol. 1999; Scholar). the that NADsyn2 NAD synthesis ammonia as an amide donor, NADsyn2 may NAD synthesis in these tissues. on a of nicotinamide mononucleotide adenylyltransferase for for NaMN (14Emanuelli M. Carnevali F. Saccucci F. Pierella F. Amici A. Raffaelli N. Magni G. J. Biol. Chem. 2001; 276: 406-412Google Scholar), NAD may also be via conversion of to NAD in the tissues. a of the of NAD biosynthesis in including on quinolinic acid phosphoribosyltransferase and nicotinic acid phosphoribosyltransferase expression in animal tissues In the present study, we identified glutamine- and ammonia-dependent human NAD synthetases, NADsyn1 and NADsyn2, with distinct tissue distribution of the synthetases, and we obtained that the domain confers glutamine on the Our results that the glutamine-dependent NAD synthetase is classified as a glutamine amidase into the nitrilase family and that the identified metabolic the ammonia-dependent NAD synthetase a role in NAD These to to catalytic activity of the and to mechanisms of cellular NAD metabolism. We K. for