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Journal of Biochemistry Advance Access originally published online on January 17, 2009
Journal of Biochemistry 2009 145(4):493-503; doi:10.1093/jb/mvp007
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Gene Identification and Characterization of 5-Formyl-3-Hydroxy-2-Methylpyridine 4-Carboxylic Acid 5-Dehydrogenase, an NAD+-Dependent Dismutase*

Nana Yokochi1,{dagger}, Yu Yoshikane1,{dagger}, Sora Matsumoto1,{dagger}, Manaho Fujisawa1, Kouhei Ohnishi2 and Toshiharu Yagi1,{ddagger}

1Department of Bioresources Science, Faculty of Agriculture; and 2Research Institute of Molecular Genetics, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan

{ddagger}To whom correspondence should be addressed. Tel: +81 88 864 5191, Fax: +81 88 864 5191, E-mail: yagito{at}kochi-u.ac.jp

Received December 6, 2008; Accepted December 26, 2008


  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 B6). The homogenously purified recombinant enzyme has a molecular mass of 59.1 kDa and is a homodimeric protein. FHMPC dehydrogenase catalyses practically irreversible oxidation (kcat = 204 s–1) of FHMPC (Km = 48.2 µM) by NAD+ (Km = 34.3 µM) to 3-hydroxy-2-methyl-pyridine 4, 5-dicarboxylic acid (HMPDC), and practically irreversible reduction (kcat = 217 s–1) of FHMPC (Km = 24.9 µM) by NADH (Km = 12.4 µM) 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.

Key Words: 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) dehydrogenase, NAD+-dependent dismutase, Mesorhizobium loti, vitamin B6-degradation pathway

Abbreviations: FHMPC, 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid; HMPDC, 3-hydroxy-2-methyl-pyridine 4, 5-dicarboxylic acid

{dagger}These authors are equal contributors to this study.


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