Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

Abstract

Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-Å resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN 3 NMN 3 NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN 3 NaAD 3 NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. genomic reconstruction ͉ in vitro reconstitution ͉ mathematical modeling ͉ NAD intermediates ͉ substrate preference

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

“…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.…”

Structural Analysis of the NH3-dependent NAD+Synthetase from Deinococcus radiodurans

“…deviation of 1.1 Å for 258 equivalent Cα positions in residues 11-281 of draNADS, a Z-score of 36.7, and a sequence identity of 59%), 6 and (iv) ftuNADS (PDB code 3FIU, r.m.s. deviation of 1.6 Å for 234 equivalent Cα positions in residues 25-272 of draNADS, a Z-score of 30.0, and a sequence identity of 36%) 9 ( Fig. 2(a)).…”

“…[5][6][7][8][9] As the prokaryotic and eukaryotic NADS differ in size, enzymatic activity and substrate requirements, NADS is an attractive target for the development of a new class of antibiotics. The NADS homolog (UniProt code Q9RYV5) in Deinococcus radiodurans encodes a protein of 287 amino acid residues, with 59% sequence identity to that of E. coli.…”

Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae

“…1a are pathogenic, there seems to be no connection between pathogenicity and having the salvage pathway involving NiaX or PnuC alone or involving both importers. Additionally, there seems to be no larger connection between pathogenicity in bacteria that contain de novo synthesis alone (Helicobacter pylori), both the de novo and salvage pathways (Bacillus anthracis), or an alternative de novo/salvage pathway (Francisella tularensis) (Huang et al, 2008;Sorci et al, 2009). The multitude of strategies utilized by these One-way ANOVA and Dunn's multiple comparison post-test were performed using TIGR4 in the related condition as the basis of comparison (n54) with no significant differences observed.…”