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Journal of Biochemistry Advance Access originally published online on January 23, 2008
Journal of Biochemistry 2008 143(5):603-609; doi:10.1093/jb/mvn010
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© 2008 The Japanese Biochemical Society.

Gene Identification and Characterization of the Pyridoxine Degradative Enzyme 4-Pyridoxic Acid Dehydrogenase from the Nitrogen-fixing Symbiotic Bacterium Mesorhizobium loti MAFF303099{dagger}

Fei Ge1, Nana Yokochi1, Yu Yoshikane1, Kouhei Ohnishi2 and Toshiharu Yagi1,*

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

*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 8, 2007; Accepted January 12, 2008


  Abstract

The gene encoding 4-pyridoxic acid dehydrogenase was identified as mlr6792 in a chromosome of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099. The enzyme is the fourth enzyme in the vitamin B6 (pyridoxine)-degradation pathway I. The recombinant enzyme with a his-tag over-expressed in Escherichia coli cells was a membrane-bound protein, and purified to homogeneity. The enzyme was a monomeric protein with a molecular weight of 59,000, and a flavoprotein containing one mole of FAD per mole of subunit. The optimum pH and temperature, and Km for 4-pyridoxic acid were pH 8.5 and 30°C, and 29 µM, respectively. The enzyme was a glucose-methanol-choline (GMC) family protein with two signature patterns, FAD-binding residues, a putative active site histidine residue and a probable transmembrane segment.

Key Words: membrane-bound enzyme, 4-pyridoxic acid dehydrogenase, pyridoxine-degradation, Mesorhizobium loti, vitamin B6

Abbreviations: KP buffer, potassium phosphate buffer; DCIP, 2, 6-dichloroindophenol; GMC, glucose-methanol-choline; FHMPC, 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic acid; HMPDC, 3-hydroxy-2-methylpyridine-4, 5-dicarboxylic acid; HMPC, 3-hydroxy-2-methylpyridine-5-carboxylic acid

{dagger}The DDBJ Accession No of 4-pyridoxic acid dehydrogenase is AB 362509.


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