Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli

Abstract

The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD.Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31 555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60306-Da protein.For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate.High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.Despite the central importance of NAD to metabolism, the biochemistry and regulation of its de novo formation are still not completely clarified. In all known organisms examined to date the pyridine ring of the NAD molecule is synthesized via quinolinate (pyridine-2,3-dicarboxylate) ; quinolinate formation occurs via four different pathways depending on the organism [l -31.In Escherichia coli and Salmonella typhimurium quinolinate biosynthesis was suggested to be catalyzed by a quinolinate synthetase complex [4] from L-aspartate and dihydroxyacetone phosphate [2, 31. Up to six intermediates have been postulated for the conversion of L-aspartate and dihydroxyacetone phosphate into quinolinate [2], an exact reaction sequence, however, has not been established yet.The quinolinate synthetase complex consists of the two enzymes quinolinate synthetases A and B, which are the gene products of genes nadA and nadB. Quinolinate synthetase B has been identified and characterized as FAD-dependent L-aspartate oxidase [5]. It catalyses oxidation of L-aspartate to iminoaspartate which is condensed with dihydroxyacetone phosphate to quinolinate under the action of quinolinate synthetase A (Fig. 1). Correspondence to H. G. Quinolinate is further converted to nicotinic acid mononucleotide via decarboxylation and phosphoribosylation by the nadC gene product quinolinate phosphoribosyltransferase [6] (Fig. 1). nadC mutant strains are not able to convert and thus excrete quinolinate into the medium.The three structural genes, nadA, nadB and nadC have been located at 17 min, 56 min [7] and 1.5 min [8], respectively, on the E. coli genetic linkage map [9]; very similar locations (17 min, 55 min and 3 min respectively) were identified [lo] on the S. typhimurium genetic linkage map.In order to investigate the molecular biology of quinolinate biosynthesis we have cloned and sequen...

Carbon monoxide-induced activation of gene expression in Rhodospirillum rubrum requires the product of cooA, a member of the cyclic AMP receptor protein family of transcriptional regulators

“…The sequence predicts that the 2.5-kb fragment does not contain the entire nadB gene. No start codon was detected for the gene, and the predicted protein is 230 to 240 residues shorter at the N terminus than are its E. coli (18) and Bacillus subtilis (49) homologs, suggesting that approximately half the gene is present on the sequenced fragment. This also suggests that the promoter for these nad genes is not present on this fragment.…”

“…The upstream gene, nadB (Fig. 1D), is 43% identical (58% similar) to nadB of E. coli (18), while the downstream gene, nadC, is 31% identical (51% similar) to nadC from E. coli (20). In E. coli and other bacteria, NadB (Laspartate oxidase) and NadC (quinolinic acid phosphoribosyltransferase) catalyze reactions in the de novo biosynthesis of NAD (19,26,39).…”

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis

“…On the basis of dase, as does its counterpart in E. coli (6). The finding that B. subtilis gene nadB.…”

“…4) (35). However, examination of the amino acid sequences of the E. coli and S. typhimurium NadA proteins reveals several conserved Cys-X-X-Cys sequences (6,8).…”

“…1). In E. coli, the latter two enzymes are encoded by the unlinked genes nadB and nadA4, respectively, both of which have been cloned and sequenced (6). Qa is then converted to nicotinic acid mononucleotide (NaMN) by Qa phosphoribosyltransferase, which is coded for by the nadC gene (23).…”