Gene identification and characterization of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD+-dependent dismutase

J Biochem. 2009 Apr;145(4):493-503. doi: 10.1093/jb/mvp007. Epub 2009 Jan 17.

Abstract

A chromosomal gene, mlr6793, in Mesorhizobium loti was identified as the gene encoding 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) dehydrogenase (dismutase) involved in the degradation pathway for pyridoxine (vitamin B(6)). The homogenously purified recombinant enzyme has a molecular mass of 59.1 kDa and is a homodimeric protein. FHMPC dehydrogenase catalyses practically irreversible oxidation (k(cat) = 204 s(-1)) of FHMPC (K(m) = 48.2 microM) by NAD(+) (K(m) = 34.3 microM) to 3-hydroxy-2-methyl-pyridine 4, 5-dicarboxylic acid (HMPDC), and practically irreversible reduction (k(cat) = 217 s(-1)) of FHMPC (K(m) = 24.9 microM) by NADH (K(m) = 12.4 microM) to 4-pyridoxic acid. When the enzyme reaction was started with the combination of FHMPC and NAD(+) or that of FHMPC and NADH, HMPDC and 4-pyridoxic acid were produced in an almost equimolar ratio throughout the reaction. FHMPC dehydrogenase belongs to the 3-hydroxyacyl-CoA dehydrogenase family with 31% identity with the human enzyme: it has probable catalytic diad residues, i.e. His137 and Glu149. The H137L mutant enzyme showed no measurable activity. The E149Q one was stable in contrast to the corresponding human 3-hydroxyacyl-CoA dehydrogenase mutant, and showed unique pH optima depending on the co-substrates used for the reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / genetics*
  • Alcohol Oxidoreductases / isolation & purification
  • Amino Acid Sequence
  • Biocatalysis
  • Cloning, Molecular
  • Electrophoresis, Agar Gel
  • Electrophoresis, Polyacrylamide Gel
  • Genes, Bacterial*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Multigene Family
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • NAD / metabolism*
  • Phylogeny
  • Pyridoxine / chemistry
  • Pyridoxine / metabolism
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Rhizobium / cytology
  • Rhizobium / enzymology*
  • Rhizobium / genetics*
  • Rhizobium / growth & development
  • Sequence Homology, Amino Acid

Substances

  • Mutant Proteins
  • Recombinant Proteins
  • NAD
  • Alcohol Oxidoreductases
  • 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic acid dehydrogenase
  • Pyridoxine

Associated data

  • GENBANK/AB362565