The gene (mlr6788) of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 has been identified as a gene coding for 2-methyl-3-hydroxypyridine-5-carboxylic acid dioxygenase (MHPCO), the seventh enzyme in degradation pathway I for pyridoxine, a free form of vitamin B(6). The gene was cloned and overexpressed in Escherichia coli cells co-transformed with chaperonin genes. The homogeneous recombinant enzyme showed similar enzymatic properties to the enzyme from Pseudomonas sp. MA-1. MHPCO was essential for the assimilation of pyridoxine in M. loti, but not for its growth in a nutrient-rich medium. From the infection experiment of a symbiotic plant Lotus japonicus with an M. loti mlr6788 gene disruptant, MHPCO was demonstrated to be dispensable for at least nodule formation on roots of seedlings in symbiosis.