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 ammoniadependent 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 ammoniadependent 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.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 calciummobilizing agents such as cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (1) and serves as a substrate for post-translational modifications of protein, mono-(2-4) and poly(ADP-ribosyl)ations (5). Depletion of cellular NAD by poly-(ADP-ribosyl)transferase activation in response to DNA damage results in cell death (6). Increased NAD synthesis has been shown to extend life span in yeast (7) and in Caenorhabditis elegans (8) via activation of an NAD-dependent histone deacetylase, silent information regulator 2 (Sir2) (9). 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 (10). Recent publications have demonstrated that fluctuation of the NAD level in cells seems to have significant impact on their physiology. Despite the...

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A rat T-cell antigen RT6.1 catalyzes NAD glycohydrolysis but not ADP-ribose transfer, even though the antigen has significant amino acid identity with eucaryotic arginine-specific ADP-ribosyltransferases. Since a highly conserved Glu in the catalytic region of these transferases is substituted with Gln at position 207 in RT6.1, we replaced the Gln with Glu, Asp, or Ala, by site-directed mutagenesis. The Glu-207 mutant produced ADP-ribosylarginine during incubation with NAD and L-arginine. The Asp-207 mutant but not the Ala-207 mutant produced ADP-ribosylarginine, but at a lower rate. In contrast, these mutations affected NAD glycohydrolase activity of RT6.1 to a much lesser extent. Kinetic studies of transferase reaction revealed that k cat of the Glu-207 mutant increased compared to findings with the Asp-207 mutant. Moreover, the mouse homologue of rat RT6 lost arginine-specific ADP-ribosyltransferase activity when Glu-207 was replaced with Gln. Thus, Glu-207 in rodent T-cell RT6 antigens is essential for transfer reaction of ADP-ribose to arginine.Arginine-specific ADP-ribosyltransferase catalyzes transfer of the ADP-ribose moiety of NAD to simple guanidino compounds such as arginine or an arginine residue of a target protein, forming ADP-ribose-acceptor adducts (1, 2). Molecular cloning has revealed the primary structures of ADP-ribosyltransferases in eucaryotes, including rabbit (3) and human skeletal muscles (4) and chicken bone marrow cells (5). Homology searches revealed highly conserved regions in the deduced amino acid sequences of chicken and skeletal muscle transferases (3-5). A gene with overall sequence similarity to these transferases was cloned from chicken erythroblasts; the functional expression was not described (6).The only known protein to which these arginine-specific ADP-ribosyltransferases show significant homology is the rat (7,8) and mouse (9) T-cell antigenic system RT6 (3, 5). It has been reported that RT6-specific antisera activate T-cells (10) and that defects in RT6 expression are associated with the pathogenesis of autoimmune insulin-dependent diabetes in diabetes-prone BB rats (11). Based on sequence similarity, Takada et al. (12) examined enzyme activities of the rat RT6.2 expressed in mammary adenocarcinoma cells and found that cells transfected with the RT6.2 gene exhibited NAD glycohydrolase (NADase), but not ADP-ribosyltransferase, activity. In contrast, Haag et al. (13) detected an arginine-specific auto-ADP-ribosylation of RT6.2 but no modification on RT6.1. More recently, Maehama et al. (14) have reported that RT6.1 was auto-ADP-ribosylated at arginine residues. Thus, whether rat RT6 antigens are indeed arginine-specific ADP-ribosyltransferases has remained controversial.We report here that the mutant RT6.1 in which Gln-207 was replaced with glutamic acid exhibited arginine-specific ADPribosyltransferase activity, while wild-type RT6.1 exhibited only NADase activity. Furthermore, the mouse homologue of rat RT6 (MRT6H), a recently characterized arginine-specific ADP-ribo...

We have reported the purification and characterization of arginine-specific ADP-ribosyltransferase from hen liver nuclei [Tanigawa, Y. et al. (1984) J. Biol. Chem. 259, 2022-2029] and the DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in the nuclei [Mishima, K. et al. (1989) Eur. J. Biochem. 179, 267-273]. In the present study, we obtained evidence that among various tissues and cells from chicken, polymorphonuclear cells, so-called heterophils, possess both the ADP-ribosyltransferase and p33 at high levels. Percoll density gradient centrifugation of the postnuclear fraction of the heterophils revealed the co-localization of ADP-ribosyltransferase with p33 in the granule fraction. The enzyme and p33 were purified approximately 219- and 3.77-fold, respectively, from postnuclear pellet fraction to apparent homogeneity. The properties of heterophil ADP-ribosyltransferase and p33 were compared with those of the liver enzyme and p33. The molecular mass of the heterophil enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 27.5 kDa. The enzyme activity was stimulated by a sulfhydryl agent and inhibited by lysolecithin, NaCl, and inorganic phosphate. The mono(ADP-ribosyl)ation of p33 was markedly enhanced by polyanion, such as DNA, RNA, or poly(L-glutamate). SDS-polyacrylamide gel electrophoretic analysis after limited trypsin proteolysis of p33s, purified from chicken heterophils and liver, showed much the same pattern. Thus, it appears that ADP-ribosyltransferase and p33 present in heterophils are identical to those in the liver, respectively. p33 is considered to be an in situ substrate for ADP-ribosyltransferase, since it was specifically mono(ADP-ribosyl)ated in permeabilized heterophils.(ABSTRACT TRUNCATED AT 250 WORDS)

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