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 (K(m)) of 385 muM for pyridoxine and pyridoxal 5'-phosphates, but its specific constant [maximum velocity (V(max))/K(m)] was nearly 2.5 times higher for the former than for the latter.
Tazoe
Masaaki
M
Department of Applied Microbiology, Nippon Roche Research Center, Kanagawa, Japan. m.tazoe@lab.tamagawa,ac,jp
Ichikawa
Keiko
K
Hoshino
Tatsuo
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D003593:Cytoplasm / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D011736:Pyridoxine / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000201:enzymology*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003470:Culture Media; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; C027138:pyridoxine phosphate phosphatase; D010744:Phosphoric Monoester Hydrolases; D011736:Pyridoxine; C010627:pyridoxamine phosphate