Genomics-driven Reconstruction of Acinetobacter NAD Metabolism

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.Acinetobacter baumannii is an emerging pathogen that belongs to a relatively underexplored branch of ␥-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 (1, 2), 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 (3-7). 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 (8, 9). 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) 4 of the NadD family and NAD synthetase of the NadE family are widely recognized as the most promising...

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

“…Other than the classical Preiss-Handler pathway outlined above, alternative pathways for salvage of NAD exist in a number of lineages. Known examples include the relatively rare non-deamidating routes, like the salvage of nicotinamide in F. tularensis that is performed by combined activity of a nicotinamide phosphoribosyltransferase (nadV) and a NMN adenylyltransferase (NadM, in some lineages is also a NaMN adenyltransferase), 12,52 or the salvage of ribosylnicotinamide (RNm) by the bifunctional N-ribosylnicotinamide kinase/NMN adenylyltransferase NadR and its associated RNm transporter PnuC 13,53 found in enterobacteriaceae and some other bacteria. In F. tularensis, the enzyme NMN synthetase, a paralog of NadE, provides a unique path for de novo synthesis of NAD from tryptophan in the absence of NadD via the amidation of NaMN.…”

“…12,13,[50][51][52][53][54][55] While there is diversity in the NAD-centered pyridine nucleotide cycle across bacteria, especially in terms of the different routes of salvage and overlap with de novo synthesis, the following key enzymes have been recognized as key players in the pathway ( Fig. 1): 12,13,[50][51][52][53][54][55] (1) a multi-domain carboxylating nicotinate/ quinolinate phosphoribosyltransferase (nadC in Fig. 1) capable of synthesizing nicotinate monophosphate ribonucleotide (NaMN) from quinolinic acid during de novo synthesis of NAD; (2) the PncA nicotinamidase that initiates the classical salvage arm of the pathway by catalyzing the hydrolysis of free nicotinamide (NM) to nicotinate (NaM); (3) the nicotinate phosphoribosyltransferase pncB, a TIM barrel enzyme of the NAPRTase family, that transfers a phosphoribosyl moiety to nicotinate thus generating nicotinate D-ribonucleotide (NaMN); (4) a HIGH nucleotidyltransferase (NadD) that adenylates the nucleotide formed in the prior steps using ATP to form the dinucleotide; (5) a PP-loop fold NAD synthase (NadE) that adenylates the carboxyl group of nicotinate using ATP and subsequently ligates it to ammonia to form a nicotinamide moiety.…”

“…12,13,[50][51][52][53][54][55] While there is diversity in the NAD-centered pyridine nucleotide cycle across bacteria, especially in terms of the different routes of salvage and overlap with de novo synthesis, the following key enzymes have been recognized as key players in the pathway ( Fig. 1): 12,13,[50][51][52][53][54][55] (1) a multi-domain carboxylating nicotinate/ quinolinate phosphoribosyltransferase (nadC in Fig.…”

“…Gene context analysis reveals that the paralog involved in NAD synthesis is typically distinguished by its routine occurrence in operons encoding other NAD salvage/biosynthesis enzymes such as the nicotinate phosphoribosyltransferase, adenylyl transferase, Nudix ADPribose pyrophosphatase, glutamine synthetase and its regulatory protein PII. 12,13,50,52,53 However, the second paralog in these organisms displays a very distinct gene neighborhood context that never contains conventional NAD salvage/biosynthesis genes (Fig. 5).…”

Acinetobacter baylyi ADP1: Transforming the choice of model organism

“…Genome annotation, single-gene deletion mutants, enzyme kinetics, and chromosomal expression of heterologous genes revealed alternative NAD metabolism which showed that current drug targets, NadD and NadE, are absent or dispensable in A. baylyi (48). Thus, NadM, which is unconditionally essential in A. baylyi, is proposed as an alternative drug target for the multidrug-resistant pathogen A. baumannii (48,49). Similar approaches were used to predict and validate a pathway to consume D-glucarate, including its regulatory mechanisms (50).…”