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

• Published in: Bioscience, biotechnology, and biochemistry Vol. 69; no. 12; pp. 2277 - 2284
• Main Authors: Tazoe, M.(Nippon Roche Research Center, Kamakura, Kanagawa (Japan)), Ichikawa, K, Hoshino, T
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.12.2005; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Amino Acid Sequence; BACTERIA FIJADORA DEL NITROGENO; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; BACTERIE FIXATRICE DE L'AZOTE; Biological and medical sciences; Cations, Divalent - chemistry; Cations, Divalent - metabolism; CHEMICOPHYSICAL PROPERTIES; CHROMATOGRAPHIE EN COLONNE; Chromatography, Ion Exchange; COLUMN CHROMATOGRAPHY; CROMATOGRAFIA DE COLUMNA; Culture Media; Cytoplasm - enzymology; ELECTROFORESIS; ELECTROPHORESE; ELECTROPHORESIS; ENZIMAS; ENZYME; ENZYMES; FOSFATOS; Fundamental and applied biological sciences. Psychology; Kinetics; METAL; METALES; METALS; Molecular Weight; NITROGEN FIXING BACTERIA; phosphatase; PHOSPHATE; PHOSPHATES; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - isolation & purification; Phosphorylation; PIRIDOXINA; PRODUCTOS; PRODUCTS; PRODUIT; PROPIEDADES FISICOQUIMICAS; PROPRIETE PHYSICOCHIMIQUE; PURIFICACION; PURIFICATION; Pyridoxal Phosphate - metabolism; Pyridoxamine - analogs & derivatives; Pyridoxamine - metabolism; PYRIDOXINE; Pyridoxine - metabolism; pyridoxine 5′-phosphate phosphatase; Sinorhizobium (Rhizobium) meliloti; Sinorhizobium meliloti; Sinorhizobium meliloti - enzymology; vitamin B; Phosphates; Purification; Enzyme; Sinorhizobium (Rhizobium) meliloti; Vitamin; Phosphoric monoester hydrolases; pyridoxine 5'-phosphate phosphatase; vitamin B6; Esterases; Pyridoxine; Characterization; Bacteria; Hydrolases; phosphatase; Rhizobiaceae; Sinorhizobium meliloti
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.69.2277

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 5f-phosphate and other physiologically important phosphorylated compounds. The enzyme had the same Michaelis constant (Km) of 385 ILM for pyridoxine and pyridoxal 5'-phosphates, but its specific constant [maximum velocity (Vsub(max)/Ksub(m)] was nearly 2.5 times higher for the former than for the latter.