Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB.
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.
Flachmann
R
R
Institut für Biochemie, Technische Hochschule Darmstadt, Federal Republic of Germany.
Kunz
N
N
Seifert
J
J
Gütlich
M
M
Wientjes
F J
FJ
Läufer
A
A
Gassen
H G
HG
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*
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