Gene identification and characterization of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD+-dependent dismutase.
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.
Yokochi
Nana
N
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan.
Yoshikane
Yu
Y
Matsumoto
Sora
S
Fujisawa
Manaho
M
Ohnishi
Kouhei
K
Yagi
Toshiharu
T
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D010802:Phylogeny; D011736:Pyridoxine / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012231:Rhizobium / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid
D050505:Mutant Proteins; D011994:Recombinant Proteins; D009243:NAD; D000429:Alcohol Oxidoreductases; C050710:5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic acid dehydrogenase; D011736:Pyridoxine