Purification and Characterization of Pyridoxine 5′-Phosphate Phosphatase from Sinorhizobium meliloti

Abstract

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 μM 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.