Keiko Ichikawa

Production of Vitamin B6 in Rhizobium

The production of vitamin B6 was studied in about 1,590 bacterial isolates from soil, and an isolate, 28-21, identified as Rhizobium leguminosarum was obtained as a vitamin B6 high producer. Then, the production of vitamin B6 by commercially available Rhizobium strains was examined, and many of the tested strains excreted large amounts of vitamin B6 into the culture broth. The best producer of vitamin B6 was R. meliloti IFO 14782, which produced 51 mg per liter. Media study for the vitamin B6 production was done with R. meliloti IFO 14782; the strain was able to excrete 84 mg of vitamin B6 per liter, 79 mg per liter of which was pyridoxol.

Flavin Adenine Dinucleotide-Dependent 4-Phospho-d-Erythronate Dehydrogenase Is Responsible for the 4-Phosphohydroxy-l-Threonine Pathway in Vitamin B6 Biosynthesis in Sinorhizobium meliloti

The vitamin B6 biosynthetic pathway in Sinorhizobium meliloti is similar to that in Escherichia coli K-12; in both organisms this pathway includes condensation of two intermediates, 1-deoxy-D-xylulose 5-phosphate and 4-phosphohydroxy-L-threonine (4PHT). Here, we report cloning of a gene designated pdxR that functionally corresponds to the pdxB gene of E. coli and encodes a dye-linked flavin adenine dinucleotide-dependent 4-phospho-D-erythronate (4PE) dehydrogenase. This enzyme catalyzes the oxidation of 4PE to 3-hydroxy-4-phosphohydroxy-alpha-ketobutyrate and is clearly different in terms of cofactor requirements from the pdxB gene product of E. coli, which is known to be an NAD-dependent enzyme. Previously, we revealed that in S. meliloti IFO 14782, 4PHT is synthesized from 4-hydroxy-l-threonine and that this synthesis starts with glycolaldehyde and glycine. However, in this study, we identified a second 4PHT pathway in S. meliloti that originates exclusively from glycolaldehyde (the major pathway). Based on the involvement of 4PE in the 4PHT pathway, the incorporation of different samples of 13C-labeled glycolaldehyde into pyridoxine molecules was examined using 13C nuclear magnetic resonance spectroscopy. On the basis of the spectral analyses, the synthesis of 4PHT from glycolaldehyde was hypothesized to involve the following steps: glycolaldehyde is sequentially metabolized to D-erythrulose, D-erythrulose 4-phosphate, and D-erythrose 4-phosphate by transketolase, kinase, and isomerase, respectively; and D-erythrose 4-phosphate is then converted to 4PHT by the conventional three-step pathway elucidated in E. coli, although the mechanism of action of the enzymes catalyzing the first two steps is different.

Biosynthesis of vitamin B6 in Rhizobium: in vitro synthesis of pyridoxine from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine.

Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5′-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.

Purification and characterization of pyridoxine 5'-phosphate phosphatase from Sinorhizobium meliloti.

Here we report the purification and biochemical characterization of a pyridoxine 5′-phosphate phosphatase involved in the biosynthesis of pyridoxine in Sinorhizobium meliloti. The phosphatase was localized in the cytoplasm and purified to electrophoretic homogeneity by a combination of EDTA/lysozyme treatment and five chromatography steps. Gel-filtration chromatography with Sephacryl S-200 and SDS/PAGE demonstrated that the protein was a monomer with a molecular size of approximately 29 kDa. The protein required divalent metal ions for pyridoxine 5′-phosphate phosphatase activity, and specifically catalyzed the removal of Pi from pyridoxine and pyridoxal 5′-phosphates at physiological pH (about 7.5). It was inactive on pyridoxamine 5′-phosphate and other physiologically important phosphorylated compounds. The enzyme had the same Michaelis constant (Km) of 385 μM for pyridoxine and pyridoxal 5′-phosphates, but its specific constant [maximum velocity (Vmax)/Km] was nearly 2.5 times higher for the former than for the latter.