title: Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB.
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 60,306-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.
authors.affiliation: Institut für Biochemie, Technische Hochschule Darmstadt, Federal Republic of Germany.
authors.forename: R
authors.initials: R
authors.lastname: Flachmann
authors.forename: N
authors.initials: N
authors.lastname: Kunz
authors.forename: J
authors.initials: J
authors.lastname: Seifert
authors.forename: M
authors.initials: M
authors.lastname: Gütlich
authors.forename: F J
authors.initials: FJ
authors.lastname: Wientjes
authors.forename: A
authors.initials: A
authors.lastname: Läufer
authors.forename: H G
authors.initials: HG
authors.lastname: Gassen
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009243:NAD / Q000096:biosynthesis*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011725:Pyridines / Q000378:metabolism*; D017378:Quinolinic Acid; D011805:Quinolinic Acids / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011725:Pyridines; D011805:Quinolinic Acids; D009243:NAD; D000594:Amino Acid Oxidoreductases; C031955:L-aspartate oxidase, E coli; D004262:DNA Restriction Enzymes; D017378:Quinolinic Acid