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• W2076184445 abstract "Enzymes involved in the last steps of NAD biogenesis, nicotinate mononucleotide adenylyltransferase (NadD) and NAD synthetase (NadE), are conserved and essential in most bacterial species and are established targets for antibacterial drug development. Our genomics-based reconstruction of NAD metabolism in the emerging pathogen Acinetobacter baumannii revealed unique features suggesting an alternative targeting strategy. Indeed, genomes of all analyzed Acinetobacter species do not encode NadD, which is functionally replaced by its distant homolog NadM. We combined bioinformatics with genetic and biochemical techniques to elucidate this and other important features of Acinetobacter NAD metabolism using a model (nonpathogenic) strain Acinetobacter baylyi sp. ADP1. Thus, a comparative kinetic characterization of PncA, PncB, and NadV enzymes allowed us to suggest distinct physiological roles for the two alternative, deamidating and nondeamidating, routes of nicotinamide salvage/recycling. The role of the NiaP transporter in both nicotinate and nicotinamide salvage was confirmed. The nondeamidating route was shown to be transcriptionally regulated by an ADP-ribose-responsive repressor NrtR. The NadM enzyme was shown to possess dual substrate specificity toward both nicotinate and nicotinamide mononucleotide substrates, which is consistent with its essential role in all three routes of NAD biogenesis, de novo synthesis as well as the two salvage pathways. The experimentally confirmed unconditional essentiality of nadM provided support for the choice of the respective enzyme as a drug target. In contrast, nadE, encoding a glutamine-dependent NAD synthetase, proved to be dispensable when the nondeamidating salvage pathway functioned as the only route of NAD biogenesis. Enzymes involved in the last steps of NAD biogenesis, nicotinate mononucleotide adenylyltransferase (NadD) and NAD synthetase (NadE), are conserved and essential in most bacterial species and are established targets for antibacterial drug development. Our genomics-based reconstruction of NAD metabolism in the emerging pathogen Acinetobacter baumannii revealed unique features suggesting an alternative targeting strategy. Indeed, genomes of all analyzed Acinetobacter species do not encode NadD, which is functionally replaced by its distant homolog NadM. We combined bioinformatics with genetic and biochemical techniques to elucidate this and other important features of Acinetobacter NAD metabolism using a model (nonpathogenic) strain Acinetobacter baylyi sp. ADP1. Thus, a comparative kinetic characterization of PncA, PncB, and NadV enzymes allowed us to suggest distinct physiological roles for the two alternative, deamidating and nondeamidating, routes of nicotinamide salvage/recycling. The role of the NiaP transporter in both nicotinate and nicotinamide salvage was confirmed. The nondeamidating route was shown to be transcriptionally regulated by an ADP-ribose-responsive repressor NrtR. The NadM enzyme was shown to possess dual substrate specificity toward both nicotinate and nicotinamide mononucleotide substrates, which is consistent with its essential role in all three routes of NAD biogenesis, de novo synthesis as well as the two salvage pathways. The experimentally confirmed unconditional essentiality of nadM provided support for the choice of the respective enzyme as a drug target. In contrast, nadE, encoding a glutamine-dependent NAD synthetase, proved to be dispensable when the nondeamidating salvage pathway functioned as the only route of NAD biogenesis. IntroductionAcinetobacter baumannii is an emerging pathogen that belongs to a relatively underexplored branch of γ-proteobacteria. It can cause severe pneumonia and infections of the urinary tract, bloodstream, and other parts of the body. Some isolates of A. baumannii display resistance to many known antibiotics (1Vila J. Martí S. Sánchez-Céspedes J. J. Antimicrob. Chemother. 2007; 59: 1210-1215Crossref PubMed Scopus (311) Google Scholar, 2Dijkshoorn L. Nemec A. Seifert H. Nat. Rev. Microbiol. 2007; 5: 939-951Crossref PubMed Scopus (1306) Google Scholar), emphasizing the importance of pursuing new therapeutic targets for drug development. Biogenesis of nicotinamide adenine nucleotide (NAD), an indispensable cofactor involved in a multitude of biochemical transformations in metabolic networks of all species, was recently established as a target pathway for the development of new antibiotics (3Vilchèze C. Weinrick B. Wong K.W. Chen B. Jacobs Jr., W.R. Mol. Microbiol. 2010; 76: 365-377Crossref PubMed Scopus (45) Google Scholar, 4Huang N. Kolhatkar R. Eyobo Y. Sorci L. Rodionova I. Osterman A.L. Mackerell A.D. Zhang H. J. Med. Chem. 2010; 53: 5229-5239Crossref PubMed Scopus (23) Google Scholar, 5Sorci L. Pan Y. Eyobo Y. Rodionova I. Huang N. Kurnasov O. Zhong S. MacKerell Jr., A.D. Zhang H. Osterman A.L. Chem. Biol. 2009; 16: 849-861Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Osterman A.L. Begley T.P. Prog. Drug Res. 2007; 64 (133–170): 131Crossref PubMed Scopus (32) Google Scholar, 7Gerdes S.Y. Scholle M.D. D'Souza M. Bernal A. Baev M.V. Farrell M. Kurnasov O.V. Daugherty M.D. Mseeh F. Polanuyer B.M. Campbell J.W. Anantha S. Shatalin K.Y. Chowdhury S.A. Fonstein M.Y. Osterman A.L. J. Bacteriol. 2002; 184: 4555-4572Crossref PubMed Scopus (241) Google Scholar). Beyond its main function as a redox cofactor, NAD is consumed as a co-substrate by a number of nonredox enzymes such as bacterial DNA ligase and protein deacetylase of the CobB/Sir2 family (8Gazzaniga F. Stebbins R. Chang S.Z. McPeek M.A. Brenner C. Microbiol. Mol. Biol. Rev. 2009; 73: 529-541Crossref PubMed Scopus (139) Google Scholar, 9Sorci L. Kurnasov O. Rodionov D.A. Osterman A.L. Lew M. Hung-Wen L. Comprehensive Natural Products II. Elsevier, Oxford2010: 213-257Crossref Google Scholar). A degradative consumption of NAD by these and, likely, other (not fully elucidated) enzymes demands continuous replenishing of the NAD pool, providing further rationale for targeting essential enzymes involved in its biogenesis and recycling. Among these enzymes, nicotinate mononucleotide adenylyltransferase (NaMNAT) 4The abbreviations used are: NaMNATnicotinate mononucleotide adenylyltransferaseNaAD(P)nicotinate adenine dinucleotide (phosphate)NmnicotinamideNanicotinateNaMNnicotinate mononucleotideADPRADP-ribosePRPPphosphoribosyl pyrophosphate. of the NadD family and NAD synthetase of the NadE family are widely recognized as the most promising drug targets (6Osterman A.L. Begley T.P. Prog. Drug Res. 2007; 64 (133–170): 131Crossref PubMed Scopus (32) Google Scholar, 7Gerdes S.Y. Scholle M.D. D'Souza M. Bernal A. Baev M.V. Farrell M. Kurnasov O.V. Daugherty M.D. Mseeh F. Polanuyer B.M. Campbell J.W. Anantha S. Shatalin K.Y. Chowdhury S.A. Fonstein M.Y. Osterman A.L. J. Bacteriol. 2002; 184: 4555-4572Crossref PubMed Scopus (241) Google Scholar). First, small-molecule inhibitors targeting bacterial NadD and NadE enzymes and showing antibacterial activity were recently reported by several research groups (4Huang N. Kolhatkar R. Eyobo Y. Sorci L. Rodionova I. Osterman A.L. Mackerell A.D. Zhang H. J. Med. Chem. 2010; 53: 5229-5239Crossref PubMed Scopus (23) Google Scholar, 5Sorci L. Pan Y. Eyobo Y. Rodionova I. Huang N. Kurnasov O. Zhong S. MacKerell Jr., A.D. Zhang H. Osterman A.L. Chem. Biol. 2009; 16: 849-861Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Moro W.B. Yang Z. Kane T.A. Zhou Q. Harville S. Brouillette C.G. Brouillette W.J. J. Comb. Chem. 2009; 11: 617-625Crossref PubMed Scopus (17) Google Scholar, 11Moro W.B. Yang Z. Kane T.A. Brouillette C.G. Brouillette W.J. Bioorg. Med. Chem. Lett. 2009; 19: 2001-2005Crossref PubMed Scopus (27) Google Scholar, 12Boshoff H.I. Xu X. Tahlan K. Dowd C.S. Pethe K. Camacho L.R. Park T.H. Yun C.S. Schnappinger D. Ehrt S. Williams K.J. Barry 3rd, C.E. J. Biol. Chem. 2008; 283: 19329-19341Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 13Velu S.E. Mou L. Luan C.H. Yang Z.W. DeLucas L.J. Brouillette C.G. Brouillette W.J. J. Med. Chem. 2007; 50: 2612-2621Crossref PubMed Scopus (16) Google Scholar, 14Plata G. Hsiao T.L. Olszewski K.L. Llinás M. Vitkup D. Mol. Syst. Biol. 2010; 6: 408Crossref PubMed Scopus (107) Google Scholar). Second, our recent genomic survey of NAD metabolism (captured as “NAD(P) biosynthesis” subsystem in the SEED data base (15Overbeek R. Begley T. Butler R.M. Choudhuri J.V. Chuang H.Y. Cohoon M. de Crécy-Lagard V. Diaz N. Disz T. Edwards R. Fonstein M. Frank E.D. Gerdes S. Glass E.M. Goesmann A. Hanson A. Iwata-Reuyl D. Jensen R. Jamshidi N. Krause L. Kubal M. Larsen N. Linke B. McHardy A.C. Meyer F. Neuweger H. Olsen G. Olson R. Osterman A. Portnoy V. Pusch G.D. Rodionov D.A. Rückert C. Steiner J. Stevens R. Thiele I. Vassieva O. Ye Y. Zagnitko O. Vonstein V. Nucleic Acids Res. 2005; 33: 5691-5702Crossref PubMed Scopus (1470) Google Scholar)) confirmed that these two enzymes are conserved in the overwhelming majority of >800 diverse bacterial species with completely sequenced genomes (9Sorci L. Kurnasov O. Rodionov D.A. Osterman A.L. Lew M. Hung-Wen L. Comprehensive Natural Products II. Elsevier, Oxford2010: 213-257Crossref Google Scholar, 16Sorci L. Martynowski D. Rodionov D.A. Eyobo Y. Zogaj X. Klose K.E. Nikolaev E.V. Magni G. Zhang H. Osterman A.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3083-3088Crossref PubMed Scopus (53) Google Scholar). NadD and NadE enzymes together comprise a two-step conversion of the committed precursor nicotinic acid mononucleotide (NaMN) to NAD. Alternative de novo and salvage routes leading to NaMN synthesis converge at this nearly universal downstream pathway, explaining the conservation and essentiality of nadD and nadE genes. Nevertheless, a few groups of bacteria (including Acinetobacter spp.) appear to deviate from a common pattern lacking orthologs for one or both of these genes and pointing to the existence of alternative routes of NAD biogenesis as well as to the necessity of alternative targeting strategies. Thus, obligate intracellular pathogens Chlamydia and Rickettsia have lost the entire NAD biosynthetic machinery, replacing it by a unique capability to salvage NAD from the host cell (9Sorci L. Kurnasov O. Rodionov D.A. Osterman A.L. Lew M. Hung-Wen L. Comprehensive Natural Products II. Elsevier, Oxford2010: 213-257Crossref Google Scholar). Haemophilus influenzae lacking both nadD and nadE as well as most other common genes of NAD biosynthesis is entirely dependent on a relatively rare PnuC-NadR pathway of nicotinamide riboside (so-called V-factor) salvage (17Gerlach G. Reidl J. J. Bacteriol. 2006; 188: 6719-6727Crossref PubMed Scopus (23) Google Scholar). Another alternative route of NAD biosynthesis was recently discovered in Francisella tularensis, the causative agent of tularemia or rabbit fever (16Sorci L. Martynowski D. Rodionov D.A. Eyobo Y. Zogaj X. Klose K.E. Nikolaev E.V. Magni G. Zhang H. Osterman A.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3083-3088Crossref PubMed Scopus (53) Google Scholar). In this species lacking nadD, the NaMN intermediate is first amidated to NMN by NMN synthetase, a divergent member of the NadE family. In the second step, NMN is converted to NAD by NMNAT of the NadM family (16Sorci L. Martynowski D. Rodionov D.A. Eyobo Y. Zogaj X. Klose K.E. Nikolaev E.V. Magni G. Zhang H. Osterman A.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3083-3088Crossref PubMed Scopus (53) Google Scholar, 18Huang N. Sorci L. Zhang X. Brautigam C.A. Li X. Raffaelli N. Magni G. Grishin N.V. Osterman A.L. Zhang H. Structure. 2008; 16: 196-209Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Members of this family, which also belongs to a large superfamily of HIGH nucleotidyltransferases (19Aravind L. Anantharaman V. Koonin E.V. Proteins. 2002; 48: 1-14Crossref PubMed Scopus (118) Google Scholar), are commonly present in Archaea as a functional replacement of NadD (20Raffaelli N. Pisani F.M. Lorenzi T. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1997; 179: 7718-7723Crossref PubMed Google Scholar). They are scarcely distributed in bacteria, typically as fusion proteins with ADP-ribose pyrophosphatase. In most cases they are present in addition to (rather than instead of) NadD in the context of a dispensable nicotinamide salvage/recycling pathway, which is initiated by the conversion of nicotinamide to NMN by nicotinamide phosphoribosyltransferase (NMPRT) of the NadV family (21Gerdes S.Y. Kurnasov O.V. Shatalin K. Polanuyer B. Sloutsky R. Vonstein V. Overbeek R. Osterman A.L. J. Bacteriol. 2006; 188: 3012-3023Crossref PubMed Scopus (38) Google Scholar). The recently established essential role of NadM in F. tularensis first highlighted this family as an alternative drug target in NAD biogenesis of bacterial pathogens. This choice is additionally supported by the fact that members of NadM family are only distantly homologous to their human counterparts (22Magni G. Di Stefano M. Orsomando G. Raffaelli N. Ruggieri S. Curr. Med. Chem. 2009; 16: 1372-1390Crossref PubMed Scopus (37) Google Scholar).To evaluate the last enzymatic steps of NAD biosynthesis as antibacterial targets in A. baumannii, we combined bioinformatics with biochemical and genetic techniques to systematically analyze the complete NAD metabolic subnetwork in the non-pathogenic model organism Acinetobacter baylyi (previously called Acinetobacter sp. ADP1) (23Barbe V. Vallenet D. Fonknechten N. Kreimeyer A. Oztas S. Labarre L. Cruveiller S. Robert C. Duprat S. Wincker P. Ornston L.N. Weissenbach J. Marlière P. Cohen G.N. Médigue C. Nucleic Acids Res. 2004; 32: 5766-5779Crossref PubMed Scopus (269) Google Scholar). This analysis led to the discovery of unique aspects of NAD metabolism in the Acinetobacter group providing new guidelines for antibacterial discovery efforts.DISCUSSIONTargeting essential enzymes involved in the biosynthesis of NAD(P), the indispensable redox cofactor, has been recognized as a promising strategy for the development of novel antibiotics (4Huang N. Kolhatkar R. Eyobo Y. Sorci L. Rodionova I. Osterman A.L. Mackerell A.D. Zhang H. J. Med. Chem. 2010; 53: 5229-5239Crossref PubMed Scopus (23) Google Scholar, 5Sorci L. Pan Y. Eyobo Y. Rodionova I. Huang N. Kurnasov O. Zhong S. MacKerell Jr., A.D. Zhang H. Osterman A.L. Chem. Biol. 2009; 16: 849-861Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Moro W.B. Yang Z. Kane T.A. Zhou Q. Harville S. Brouillette C.G. Brouillette W.J. J. Comb. Chem. 2009; 11: 617-625Crossref PubMed Scopus (17) Google Scholar, 12Boshoff H.I. Xu X. Tahlan K. Dowd C.S. Pethe K. Camacho L.R. Park T.H. Yun C.S. Schnappinger D. Ehrt S. Williams K.J. Barry 3rd, C.E. J. Biol. Chem. 2008; 283: 19329-19341Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). However, the rational choice of actual drug targets requires better understanding of pathways that comprise NAD metabolic subnetworks, especially in divergent and poorly explored organisms such as pathogenic species of Acinetobacter. Indeed this study revealed that among the two nearly universal target enzymes, NadD and NadE, the former is functionally replaced in Acinetobacter by a member of a distinct NadM family, whereas the latter is potentially dispensable.Our approach to the reconstruction of NAD metabolism in Acinetobacter was based on comparative genomics analysis combined with focused experimental testing and refinement of the key bionformatics predictions. The bioinformatics analysis included all Acinetobacter species with completely sequenced genomes (7 genomes were publicly available), whereas the experimental testing was performed using a nonpathogenic strain, A. baylyi ADP1. This strain provided us with an ideal model system due to its remarkable genetic versatility (27Metzgar D. Bacher J.M. Pezo V. Reader J. Döring V. Schimmel P. Marlière P. de Crécy-Lagard V. Nucleic Acids Res. 2004; 32: 5780-5790Crossref PubMed Scopus (127) Google Scholar, 45de Berardinis V. Durot M. Weissenbach J. Salanoubat M. Curr. Opin. Microbiol. 2009; 12: 568-576Crossref PubMed Scopus (41) Google Scholar) and availability of a genome-wide collection of knock-out mutants by Genoscope (25de Berardinis V. Vallenet D. Castelli V. Besnard M. Pinet A. Cruaud C. Samair S. Lechaplais C. Gyapay G. Richez C. Durot M. Kreimeyer A. Le Fèvre F. Schächter V. Pezo V. Döring V. Scarpelli C. Médigue C. Cohen G.N. Marlière P. Salanoubat M. Weissenbach J. Mol. Syst. Biol. 2008; 4: 174Crossref PubMed Scopus (224) Google Scholar). Moreover, understanding of NAD metabolism in A. baylyi is of additional importance for its biotechnological applications in biodegradation and manufacturing of biopolymers (46Gutnick D.L. Bach H. Gerischer U. Acinetobacter Molecular Biology. Caister Academic Press, Norfolk, UK2008: 231-264Google Scholar). Indeed, maintenance and regeneration of the NAD cofactor pool is a known bottleneck in many microbial fermentations (47Verho R. Londesborough J. Penttilä M. Richard P. Appl. Environ. Microbiol. 2003; 69: 5892-5897Crossref PubMed Scopus (166) Google Scholar, 48Maicas S. Ferrer S. Pardo I. Microbiology. 2002; 148: 325-332Crossref PubMed Scopus (43) Google Scholar).In silico genomic reconstruction performed in this study revealed an extensive network of pathways leading to NAD, which is conserved in all analyzed Acinetobacter spp. and includes de novo synthesis and two alternative salvage/recycling routes (Fig. 1 and Table 1). This analysis pointed us to several unique and previously unexplored features, and targeted biochemical and genetic experiments were designed to test the key bioinformatics inferences and reconcile ambiguities and possible alternative scenarios.In the upstream part of Acinetobacter NAD subnetwork, the most unusual feature is the simultaneous presence of the two alternative (and potentially competing) Nm salvage routes, deamidating (via PncA-PncB) and nondeamidating (via NadV). A widely different phylogenomic distribution of these two alternative salvage pathways and possible evolutionary implications have been previously discussed (8Gazzaniga F. Stebbins R. Chang S.Z. McPeek M.A. Brenner C. Microbiol. Mol. Biol. Rev. 2009; 73: 529-541Crossref PubMed Scopus (139) Google Scholar, 21Gerdes S.Y. Kurnasov O.V. Shatalin K. Polanuyer B. Sloutsky R. Vonstein V. Overbeek R. Osterman A.L. J. Bacteriol. 2006; 188: 3012-3023Crossref PubMed Scopus (38) Google Scholar), and they are further illustrated in supplemental Table S4.Through in vitro characterization of all respective enzymes (Table 3) and growth phenotype analysis of engineered knock-out mutants (Table 2), we have confirmed that both salvage routes are operational in Acinetobacter. Indeed, each of them could support the normal growth of A. baylyi as a sole source of pyridine nucleotides on the minimal medium supplemented with Nm. Although the biological significance of this duality is not completely clear, some experimental data obtained here allow us to speculate that the first route is predominantly responsible for the salvage of exogenous Nm (as well as Na), especially when its concentration in the environment is very low (Table 2 and Fig. 2). On the other hand, the second, abNadV-mediated route is likely to contribute largely to the recycling of endogenous Nm, which may accumulate due to degradative utilization of NAD by CobB/Sir2 NAD-dependent protein deacetylase and, possibly, by other (yet unknown) NAD-glycohydrolases. Indirect evidence in favor of this interpretation was provided by the experimentally confirmed in vivo transcriptional regulation of nadV gene by ADPR-responsive repressor abNrtR. The ability of ADPR to disrupt binding of abNrtR to the predicted operator site in the upstream of prs-nadV operon was confirmed in vitro by EMSA analysis (Fig. 3). These observations are consistent with the hypothesis that accumulation of ADPR resulting from NAD degradation may be interpreted by the cell as a signal to activate recycling of Nm. The latter is released as a byproduct of this degradation, and the abNadV-mediated nondeamidating route appears to be the most efficient way of its conversion to NAD with the help of NMNAT activity of abNadM (see below). Notably, the first gene of the prs-nadV operon encoding a paralog of PRPP synthetase may also directly contribute to Nm recycling by feeding PRPP as a second substrate for NadV reaction (Fig. 1). It is tempting to speculate that ribosyl 5-phosphate required for the synthesis of PRPP may be at least partially supplied by the hydrolysis of ADPR (supplemental Fig. S5) by the product of nudF gene (ACIAD0275 in A. baylyi) tentatively assigned an ADPR pyrophosphatase function based on homology with other characterized members of Nudix family (49McLennan A.G. Cell. Mol. Life Sci. 2006; 63: 123-143Crossref PubMed Scopus (443) Google Scholar).The remarkable plurality of the upstream pathways in the NAD subnetwork leading to the formation of pyridine nucleotide precursors (NaMN and NMN) emphasizes the importance of NAD biogenesis and homeostasis in Acinetobacter. At the same time, a demonstrated functional redundancy of these pathways (at least in laboratory conditions) argues that none of the respective enzymes would constitute a sustainable drug target. Moreover, of the two enzymes abNadM and abNadE involved in downstream conversion of these precursors to NAD, only the former is truly indispensable, as supported by combined genetic and kinetic evidences (TABLE 1, TABLE 2, TABLE 3). The abNadM is the first documented example of dual NMNAT/NaMNAT specificity among bacterial members of the NadM family. As noticed before, this family is only sparsely represented in bacteria where NadM is usually present in addition to (and not instead of) the housekeeping NadD enzyme and contributes to an optional Nm salvage/recycling together with NadV enzyme (21Gerdes S.Y. Kurnasov O.V. Shatalin K. Polanuyer B. Sloutsky R. Vonstein V. Overbeek R. Osterman A.L. J. Bacteriol. 2006; 188: 3012-3023Crossref PubMed Scopus (38) Google Scholar). In most cases these two genes, nadV and nadM, are encoded in one operon. Importantly, all previously characterized bacterial NadM enzymes have two features distinguishing them from abNadM; (i) strong (at least 50–100-fold) substrate preference for NMN over NaMN and (ii) the presence of an additional C-terminal domain harboring ADPR pyrophosphatase of Nudix family. Both enzymatic activities are relevant for Nm salvage as discussed above for A. baylyi where the ADPR pyrophosphatase activity is encoded by a remotely located nudF gene. The structural and enzymatic analysis of the two representative bacterial NadM-NudF fusion proteins did not reveal any functional interactions between the two domains that would affect the NadM substrate preference (18Huang N. Sorci L. Zhang X. Brautigam C.A. Li X. Raffaelli N. Magni G. Grishin N.V. Osterman A.L. Zhang H. Structure. 2008; 16: 196-209Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). At the same time, the NadM (and not NadD) family is universally present in nearly all Archaea where it appears to function as the housekeeping NaMNAT/NMNAT enzyme in NAD biogenesis.5 None of the analyzed archaeal genomes revealed a presence of the bacterial-like NadM-NudF fusion protein. These observations as well as characteristic variations in gene patterns associated with deamidating and nondeamidating Nm salvage/recycling routes point to a likely archaeal origin of bacterial NadM (see supplemental Table S4 and the accompanying comments suggesting a possible evolutionary scenario).Overall, the most important practical implication of this study is the identification of abNadM as an attractive target for the development of novel antibiotics against pathogenic strains of A. baumannii. This is the second case (after F. tularensis (16Sorci L. Martynowski D. Rodionov D.A. Eyobo Y. Zogaj X. Klose K.E. Nikolaev E.V. Magni G. Zhang H. Osterman A.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3083-3088Crossref PubMed Scopus (53) Google Scholar)) where a member of NadM family functionally replaces NadD (which is otherwise present in nearly all pathogenic bacteria) and constitutes an alternative drug target. In both cases, members of NadE family were shown to be dispensable (at least in laboratory conditions). The latter observation can be tentatively projected to some other bacterial species (including pathogenic strains of Burkholderia) containing the complete nondeamidating salvage pathway comprised of NMPRT (NadV family) and NMNAT (NadM or NadR family). More generally, it reflects a fundamental difference between the two types of biochemical transformations associated with the downstream pathways of NAD biosynthesis. At least one of the two adenylyl transferase reactions converting pyridine mononucleotides (NaMN or/and NMN) to respective dinucleotides (NaAD or/and NAD) is present and unconditionally essential in all life forms (including all bacterial pathogens) except for some intracellular endosymbionts. On the other hand, the ATP-dependent amidation of the carboxylate moiety of NaAD by NAD synthetase (or, rarely, of NaMN by NMN synthetase) in some species may be bypassed (as in Francisella) or even replaced (as in Haemophilus) by the nondeamidating salvage of amidated precursors (Nm or Nm-ribose, respectively). NAD metabolism of Acinetobacter analyzed in this study is another example that illustrates this fundamental principle providing important guidelines for drug targets selection and prioritization. IntroductionAcinetobacter baumannii is an emerging pathogen that belongs to a relatively underexplored branch of γ-proteobacteria. It can cause severe pneumonia and infections of the urinary tract, bloodstream, and other parts of the body. Some isolates of A. baumannii display resistance to many known antibiotics (1Vila J. Martí S. Sánchez-Céspedes J. J. Antimicrob. Chemother. 2007; 59: 1210-1215Crossref PubMed Scopus (311) Google Scholar, 2Dijkshoorn L. Nemec A. Seifert H. Nat. Rev. Microbiol. 2007; 5: 939-951Crossref PubMed Scopus (1306) Google Scholar), emphasizing the importance of pursuing new therapeutic targets for drug development. Biogenesis of nicotinamide adenine nucleotide (NAD), an indispensable cofactor involved in a multitude of biochemical transformations in metabolic networks of all species, was recently established as a target pathway for the development of new antibiotics (3Vilchèze C. Weinrick B. Wong K.W. Chen B. Jacobs Jr., W.R. Mol. Microbiol. 2010; 76: 365-377Crossref PubMed Scopus (45) Google Scholar, 4Huang N. Kolhatkar R. Eyobo Y. Sorci L. Rodionova I. Osterman A.L. Mackerell A.D. Zhang H. J. Med. Chem. 2010; 53: 5229-5239Crossref PubMed Scopus (23) Google Scholar, 5Sorci L. Pan Y. Eyobo Y. Rodionova I. Huang N. Kurnasov O. Zhong S. MacKerell Jr., A.D. Zhang H. Osterman A.L. Chem. Biol. 2009; 16: 849-861Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Osterman A.L. Begley T.P. Prog. Drug Res. 2007; 64 (133–170): 131Crossref PubMed Scopus (32) Google Scholar, 7Gerdes S.Y. Scholle M.D. D'Souza M. Bernal A. Baev M.V. Farrell M. Kurnasov O.V. Daugherty M.D. Mseeh F. Polanuyer B.M. Campbell J.W. Anantha S. Shatalin K.Y. Chowdhury S.A. Fonstein M.Y. Osterman A.L. J. Bacteriol. 2002; 184: 4555-4572Crossref PubMed Scopus (241) Google Scholar). Beyond its main function as a redox cofactor, NAD is consumed as a co-substrate by a number of nonredox enzymes such as bacterial DNA ligase and protein deacetylase of the CobB/Sir2 family (8Gazzaniga F. Stebbins R. Chang S.Z. McPeek M.A. Brenner C. Microbiol. Mol. Biol. Rev. 2009; 73: 529-541Crossref PubMed Scopus (139) Google Scholar, 9Sorci L. Kurnasov O. Rodionov D.A. Osterman A.L. Lew M. Hung-Wen L. Comprehensive Natural Products II. Elsevier, Oxford2010: 213-257Crossref Google Scholar). A degradative consumption of NAD by these and, likely, other (not fully elucidated) enzymes demands continuous replenishing of the NAD pool, providing further rationale for targeting essential enzymes involved in its biogenesis and recycling. Among these enzymes, nicotinate mononucleotide adenylyltransferase (NaMNAT) 4The abbreviations used are: NaMNATnicotinate mononucleotide adenylyltransferaseNaAD(P)nicotinate adenine dinucleotide (phosphate)NmnicotinamideNanicotinateNaMNnicotinate mononucleotideADPRADP-ribosePRPPphosphoribosyl pyrophosphate. of the NadD family and NAD synthetase of the NadE family are widely recognized as the most promising drug targets (6Osterman A.L. Begley T.P. Prog. Drug Res. 2007; 64 (133–170): 131Crossref PubMed Scopus (32) Google Scholar, 7Gerdes S.Y. Scholle M.D. D'Souza M. Bernal A. Baev M.V. Farrell M. Kurnasov O.V. Daugherty M.D. Mseeh F. Polanuyer B.M. Campbell J.W. Anantha S. Shatalin K.Y. Chowdhury S.A. Fonstein M.Y. Osterman A.L. J. Bacteriol. 2002; 184: 4555-4572Crossref PubMed Scopus (241) Google Scholar). First, small-molecule inhibitors targeting bacterial NadD and NadE enzymes and showing antibacterial activity were recently reported by several research groups (4Huang N. Kolhatkar R. Eyobo Y. Sorci L. Rodionova I. Osterman A.L. Mackerell A.D. Zhang H. J. Med. Chem. 2010; 53: 5229-5239Crossref PubMed Scopus (23) Google Scholar, 5Sorci L. Pan Y. Eyobo Y. Rodionova I. Huang N. Kurnasov O. Zhong S. MacKerell Jr., A.D. Zhang H. Osterman A.L. Chem. Biol. 2009; 16: 849-861Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Moro W.B. Yang Z. Kane T.A. Zhou Q. Harville S. Brouillette C.G. Brouillette W.J. J. Comb. Chem. 2009; 11: 617-625Crossref PubMed Scopus (17) Google Scholar, 11Moro W.B. Yang Z. Kane T.A. Brouillette C.G. Brouillette W.J. Bioorg. Med. Chem. Lett. 2009; 19: 2001-2005Crossref PubMed Scopus (27) Google Scholar, 12Boshoff H.I. Xu X. Tahlan K. Dowd C.S. Pethe K. Camacho L.R. Park T.H. Yun C.S. Schnappinger D. Ehrt S. Williams K.J. Barry 3rd, C.E. J. Biol. Chem. 2008; 283: 19329-19341Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 13Velu S.E. Mou L. Luan C.H. Yang Z.W. DeLucas L.J. Brouillette C.G. Brouillette W.J. J. Med. Chem. 2007; 50: 2612-2621Crossref PubMed Scopus (16) Google Scholar, 14Plata G. Hsiao T.L. Olszewski K.L. Llinás M. Vitkup D. Mol. Syst. Biol. 2010; 6: 408Crossref PubMed Scopus (107) Google Scholar). Second, our recent genomic survey of NAD metabolism (captured as “NAD(P) biosynthesis” subsystem in the SEED data base (15Overbeek R. Begley T. Butler R.M. Choudhuri J.V. Chuang H.Y. Cohoon M. de Crécy-Lagard V. Diaz N. Disz T. Edwards R. Fonstein M. Frank E.D. Gerdes S. Glass E.M. Goesmann A. Hanson A. Iwata-Reuyl D. Jensen R. Jamshidi N. Krause L. Kubal M. Larsen N. Linke B. McHardy A.C. Meyer F. Neuweger H. Olsen G. Olson R. Osterman A. Portnoy V. Pusch G.D. Rodionov D.A. Rückert C. Steiner J. Stevens R. Thiele I. Vassieva O. Ye Y. Zagnitko O. Vonstein V. Nucleic Acids Res. 2005; 33: 5691-5702Crossref PubMed Scopus (1470) Google Scholar)) confirmed that these two enzymes are conserved in the overwhelming majority of >800 diverse bacterial species with completely sequenced genomes (9Sorci L. Kurnasov O. Rodionov D.A. Osterman A.L. Lew M. Hung-Wen L. Comprehensive Natural Products II. Elsevier, Oxford2010: 213-257Crossref Google Scholar, 16Sorci L. Martynowski D. Rodionov D.A. Eyobo Y. Zogaj X. Klose K.E. Nikolaev E.V. Magni G. Zhang H. Osterman A.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3083-3088Crossref PubMed Scopus (53) Google Scholar). NadD and NadE enzymes together comprise a two-step conversion of the committed precursor nicotinic acid mononucleotide (NaMN) to NAD. Alternative de novo and salvage routes leading to NaMN synthesis converge at this nearly universal downstream pathway, explaining the conservation and essentiality of nadD and nadE genes. Nevertheless, a few groups of bacteria (including Acinetobacter spp.) appear to deviate from a common pattern lacking orthologs for one or both of these genes and pointing to the existence of alternative routes of NAD biogenesis as well as to the necessity of alternative targeting strategies. Thus, obligate intracellular pathogens Chlamydia and Rickettsia have lost the entire NAD biosynthetic machinery, replacing it by a unique capability to salvage NAD from the host cell (9Sorci L. Kurnasov O. Rodionov D.A. Osterman A.L. Lew M. Hung-Wen L. Comprehensive Natural Products II. Elsevier, Oxford2010: 213-257Crossref Google Scholar). Haemophilus influenzae lacking both nadD and nadE as well as most other common genes of NAD biosynthesis is entirely dependent on a relatively rare PnuC-NadR pathway of nicotinamide riboside (so-called V-factor) salvage (17Gerlach G. Reidl J. J. Bacteriol. 2006; 188: 6719-6727Crossref PubMed Scopus (23) Google Scholar). Another alternative route of NAD biosynthesis was recently discovered in Francisella tularensis, the causative agent of tularemia or rabbit fever (16Sorci L. Martynowski D. Rodionov D.A. Eyobo Y. Zogaj X. Klose K.E. Nikolaev E.V. Magni G. Zhang H. Osterman A.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3083-3088Crossref PubMed Scopus (53) Google Scholar). In this species lacking nadD, the NaMN intermediate is first amidated to NMN by NMN synthetase, a divergent member of the NadE family. In the second step, NMN is converted to NAD by NMNAT of the NadM family (16Sorci L. Martynowski D. Rodionov D.A. Eyobo Y. Zogaj X. Klose K.E. Nikolaev E.V. Magni G. Zhang H. Osterman A.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3083-3088Crossref PubMed Scopus (53) Google Scholar, 18Huang N. Sorci L. Zhang X. Brautigam C.A. Li X. Raffaelli N. Magni G. Grishin N.V. Osterman A.L. Zhang H. Structure. 2008; 16: 196-209Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Members of this family, which also belongs to a large superfamily of HIGH nucleotidyltransferases (19Aravind L. Anantharaman V. Koonin E.V. Proteins. 2002; 48: 1-14Crossref PubMed Scopus (118) Google Scholar), are commonly present in Archaea as a functional replacement of NadD (20Raffaelli N. Pisani F.M. Lorenzi T. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1997; 179: 7718-7723Crossref PubMed Google Scholar). They are scarcely distributed in bacteria, typically as fusion proteins with ADP-ribose pyrophosphatase. In most cases they are present in addition to (rather than instead of) NadD in the context of a dispensable nicotinamide salvage/recycling pathway, which is initiated by the conversion of nicotinamide to NMN by nicotinamide phosphoribosyltransferase (NMPRT) of the NadV family (21Gerdes S.Y. Kurnasov O.V. Shatalin K. Polanuyer B. Sloutsky R. Vonstein V. Overbeek R. Osterman A.L. J. Bacteriol. 2006; 188: 3012-3023Crossref PubMed Scopus (38) Google Scholar). The recently established essential role of NadM in F. tularensis first highlighted this family as an alternative drug target in NAD biogenesis of bacterial pathogens. This choice is additionally supported by the fact that members of NadM family are only distantly homologous to their human counterparts (22Magni G. Di Stefano M. Orsomando G. Raffaelli N. Ruggieri S. Curr. Med. Chem. 2009; 16: 1372-1390Crossref PubMed Scopus (37) Google Scholar).To evaluate the last enzymatic steps of NAD biosynthesis as antibacterial targets in A. baumannii, we combined bioinformatics with biochemical and genetic techniques to systematically analyze the complete NAD metabolic subnetwork in the non-pathogenic model organism Acinetobacter baylyi (previously called Acinetobacter sp. ADP1) (23Barbe V. Vallenet D. Fonknechten N. Kreimeyer A. Oztas S. Labarre L. Cruveiller S. Robert C. Duprat S. Wincker P. Ornston L.N. Weissenbach J. Marlière P. Cohen G.N. Médigue C. Nucleic Acids Res. 2004; 32: 5766-5779Crossref PubMed Scopus (269) Google Scholar). This analysis led to the discovery of unique aspects of NAD metabolism in the Acinetobacter group providing new guidelines for antibacterial discovery efforts." @default.
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