title: Studies on the biosynthesis of bialaphos (SF-1293). 8. Purification and characterization of 2-phosphinomethylmalic acid synthase from Streptomyces hygroscopicus SF-1293.
abstract: 2-Phosphinomethylmalic acid (PMM) synthase catalyzes the condensation of phosphinopyruvic acid (PPA), an analog of oxalacetic acid, and acetyl-CoA to form PMM. The enzyme was purified approximately 700-fold from a cell-free extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism, to an electrophoretically homogeneous state. The purified PMM synthase has a subunit molecular weight of 48,000 by SDS-polyacrylamide gel electrophoresis and a native molecular weight of 90,000 approximately 98,000 by gel filtration. PMM synthase was relatively unstable, showed maximum activity at pH 8.0 and 30 degrees C, and was inhibited strongly by p-chloromercuribenzoate, iodoacetamide and EDTA. Enzyme activity suppressed by EDTA was completely restored by adding Co++ or Mn++ and partially restored by addition of Ca++, Fe++ or Mg++. The specific substrates of this enzyme are PPA or oxalacetic acid in addition to acetyl-CoA. The enzyme does not catalyze the liberation of CoA from acetyl-CoA in the presence of alpha-keto acids, such as pyruvate, alpha-ketoglutarate, deamino-alpha-ketodemethylphosphinothricin or phosphonopyruvate. The condensation reaction did not take place when propionyl-CoA or butyryl-CoA was used as a substrate in place of acetyl-CoA. The Km values of the enzyme were 0.05 mM for acetyl-CoA, 0.39 mM for PPA and 0.13 mM for oxalacetate. PMM synthase is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity; unlike (R)-citrate synthase PMM synthase was not inhibited by oxygen.
authors.affiliation: Institute of Applied Microbiology, University of Tokyo, Japan.
authors.forename: K W
authors.initials: KW
authors.lastname: Shimotohno
authors.forename: H
authors.initials: H
authors.lastname: Seto
authors.forename: N
authors.initials: N
authors.lastname: Otake
authors.forename: S
authors.initials: S
authors.lastname: Imai
authors.forename: T
authors.initials: T
authors.lastname: Murakami
mesh_terms: D002850:Chromatography, Gel; D002950:Citrate (si)-Synthase / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008292:Malate Synthase / Q000302:isolation & purification*; D008293:Malates / Q000096:biosynthesis*; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000096:biosynthesis*; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification*; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism
chemical_list: D008293:Malates; D009943:Organophosphorus Compounds; C043280:2-phosphinomethylmalic acid; D002950:Citrate (si)-Synthase; D008292:Malate Synthase; C057307:2-phosphinomethylmalic acid synthase; D007652:Oxo-Acid-Lyases