title: Purification and characterization of hydroxypyruvate reductase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.
abstract: Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 degrees C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 degrees C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.
authors.affiliation: Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Izumi
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: H
authors.initials: H
authors.lastname: Kanzaki
authors.forename: S
authors.initials: S
authors.lastname: Toki
authors.forename: S S
authors.initials: SS
authors.lastname: Miyazaki
authors.forename: H
authors.initials: H
authors.lastname: Yamada
mesh_terms: D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D001419:Bacteria / Q000201:enzymology* / Q000378:metabolism; D002850:Chromatography, Gel; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D050537:Hydroxypyruvate Reductase; D007525:Isoelectric Focusing; D007700:Kinetics; D011773:Pyruvates / Q000032:analysis; D012694:Serine / Q000096:biosynthesis; D013379:Substrate Specificity
chemical_list: D011773:Pyruvates; D012694:Serine; C012375:hydroxypyruvic acid; D000429:Alcohol Oxidoreductases; D050537:Hydroxypyruvate Reductase