Enzymes catalyze the transfer of the NAD ribonucleotidyl moiety, either in

Enzymes catalyze the transfer of the NAD ribonucleotidyl moiety, either in the form of AMP or ADPribose (ADPR), to different functional groups of Title Loaded From File proteins and nucleic acids, thereby modulating their function. NAD-dependent ADP-ribosylation and deacetylation of various target proteins, as well as NAD-dependent dephosphorylation of tRNA are well known in eukaryotes. In contrast, non redox NAD-dependent processes in bacteria are still relatively unexplored. Only few bacterial NAD-consuming enzymes have been so far characterized: i) the NAD-dependent DNA ligase, which uses the AMP Title Loaded From File moiety of NAD to activate the 59-phosphate of nicked DNA ends; ii) the NAD-dependent deacetylase CobB of the Sirt2 family, which catalyzes protein deacetylation by transferring ADPR from NAD to the acetyl group, with the release of O-acetyl-ADPR [4];iii) various mono ADP-ribosyltransferases, which catalyze the covalent attachment of single ADPR units to both endogenous and host proteins to regulate their function [5,6]; iv) an ortholog of the yeast tRNA 29-phosphotransferase, able to catalyze tRNA dephosphorylation by transferring ADPR from NAD to the 29phosphate group of tRNA, with the release of 1,2 cyclic phosphate ADPR [7]. The occurrence of an intensive NAD consumption in bacteria is suggested by the remarkably rapid turnover of the intracellular NAD pool, which also emphasizes the importance of the continuous replenishing of the dinucleotide [8]. Notably, all the products of NAD consumption can contribute to its regeneration (Figure 1). In particular, the pyridine by-products, nicotinamide mononucleotide (NMN) and nicotinamide (Nm), can be recycled back to NAD through various recycling pathways that, in recent years, have begun to be elucidated in the majority of bacterial species, thanks to genomics-guided approaches [9,10,11,12]. In turn, the ADPR moiety released from ADPribosylated proteins, or deriving from O-acetyl-ADPR and 1,2 cyclic phosphate ADPR, can be further hydrolyzed by ADPR pyrophosphatases (ADPRP) of the Nudix family, yielding AMP and ribose-5-phosphate, which may be reused in NAD biogenesisCOG1058 Is a Novel Pyrophosphatase Familyvia conversion to ATP and PRPP, respectively, as already proposed in eukaryotes [13,14,15]. In this view, the occurrence in some bacterial species of a Nudix ADPRP domain fused to the enzyme NMN adenylyltransferase (NadM) provides evidence of the strict link between NAD consumption and regeneration (Figure 1) [16,17]. Very recently, combining comparative genomic analysis, metabolic pathways reconstruction, and experimental characterization, we have identified the enzyme NMN deamidase (PncC), which converts NMN to nicotinate mononucleotide (NaMN), thus channeling the mononucleotide towards the deamidated NAD biosynthetic pathway (Figure 1) [10]. This enzyme plays a key role in NMN and Nm recycling back to NAD in the majority of bacterial species. Very often, PncC is found fused to a domain of unknown function, which belongs to the family of proteins classified as COG1058 in the Clusters of Orthologous Groups database, currently annotated as “predicted nucleotide-utilizing enzyme family, related to molybdopterin-biosynthesis enzyme MoeA” (PF00994). Members of this family share a high similarity with bacterial MoeA and its eukaryotic orthologues (i.e. the Edomains of mammalian gephyrin and plant Cnx1), which are involved in the last step of Molybdenum Cofactor (MoCo) biosynthesis [18]. In particular, these enzymes b.Enzymes catalyze the transfer of the NAD ribonucleotidyl moiety, either in the form of AMP or ADPribose (ADPR), to different functional groups of proteins and nucleic acids, thereby modulating their function. NAD-dependent ADP-ribosylation and deacetylation of various target proteins, as well as NAD-dependent dephosphorylation of tRNA are well known in eukaryotes. In contrast, non redox NAD-dependent processes in bacteria are still relatively unexplored. Only few bacterial NAD-consuming enzymes have been so far characterized: i) the NAD-dependent DNA ligase, which uses the AMP moiety of NAD to activate the 59-phosphate of nicked DNA ends; ii) the NAD-dependent deacetylase CobB of the Sirt2 family, which catalyzes protein deacetylation by transferring ADPR from NAD to the acetyl group, with the release of O-acetyl-ADPR [4];iii) various mono ADP-ribosyltransferases, which catalyze the covalent attachment of single ADPR units to both endogenous and host proteins to regulate their function [5,6]; iv) an ortholog of the yeast tRNA 29-phosphotransferase, able to catalyze tRNA dephosphorylation by transferring ADPR from NAD to the 29phosphate group of tRNA, with the release of 1,2 cyclic phosphate ADPR [7]. The occurrence of an intensive NAD consumption in bacteria is suggested by the remarkably rapid turnover of the intracellular NAD pool, which also emphasizes the importance of the continuous replenishing of the dinucleotide [8]. Notably, all the products of NAD consumption can contribute to its regeneration (Figure 1). In particular, the pyridine by-products, nicotinamide mononucleotide (NMN) and nicotinamide (Nm), can be recycled back to NAD through various recycling pathways that, in recent years, have begun to be elucidated in the majority of bacterial species, thanks to genomics-guided approaches [9,10,11,12]. In turn, the ADPR moiety released from ADPribosylated proteins, or deriving from O-acetyl-ADPR and 1,2 cyclic phosphate ADPR, can be further hydrolyzed by ADPR pyrophosphatases (ADPRP) of the Nudix family, yielding AMP and ribose-5-phosphate, which may be reused in NAD biogenesisCOG1058 Is a Novel Pyrophosphatase Familyvia conversion to ATP and PRPP, respectively, as already proposed in eukaryotes [13,14,15]. In this view, the occurrence in some bacterial species of a Nudix ADPRP domain fused to the enzyme NMN adenylyltransferase (NadM) provides evidence of the strict link between NAD consumption and regeneration (Figure 1) [16,17]. Very recently, combining comparative genomic analysis, metabolic pathways reconstruction, and experimental characterization, we have identified the enzyme NMN deamidase (PncC), which converts NMN to nicotinate mononucleotide (NaMN), thus channeling the mononucleotide towards the deamidated NAD biosynthetic pathway (Figure 1) [10]. This enzyme plays a key role in NMN and Nm recycling back to NAD in the majority of bacterial species. Very often, PncC is found fused to a domain of unknown function, which belongs to the family of proteins classified as COG1058 in the Clusters of Orthologous Groups database, currently annotated as “predicted nucleotide-utilizing enzyme family, related to molybdopterin-biosynthesis enzyme MoeA” (PF00994). Members of this family share a high similarity with bacterial MoeA and its eukaryotic orthologues (i.e. the Edomains of mammalian gephyrin and plant Cnx1), which are involved in the last step of Molybdenum Cofactor (MoCo) biosynthesis [18]. In particular, these enzymes b.