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.
Highlights
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
Utilization of a canonical NMNAT for the processing of nicotinic acid mononucleotide (NaMN) intermediate generated by de novo (NadB-NadA-NadC) or deamidating salvage (PncA-PncB) routes (Fig. 1) would be extremely inefficient or require the presence of an additional enzyme, NMN synthetase, as previously shown for F. tularensis [16]
Targeting 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 [4, 5, 10, 12]
Summary
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. 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 [9] 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 Vfactor) salvage [17]. Another alternative route of NAD biosynthesis was recently discovered in Francisella tularensis, the causative agent of tularemia or rabbit fever [16]. This analysis led to the discovery of unique aspects of NAD metabolism in the Acinetobacter group providing new guidelines for antibacterial discovery efforts
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