Studies on the Biosynthesis of Bialaphos (SF-1293). 8. Purification and Characterization of 2-phosphinomethylmalic Acid Synthase From Streptomyces Hygroscopicus SF-1293

J Antibiot (Tokyo). 1988 Aug;41(8):1057-65. doi: 10.7164/antibiotics.41.1057.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Gel
  • Citrate (si)-Synthase / isolation & purification
  • Hydrogen-Ion Concentration
  • Malate Synthase / isolation & purification*
  • Malates / biosynthesis*
  • Molecular Weight
  • Organophosphorus Compounds / biosynthesis*
  • Oxo-Acid-Lyases / isolation & purification*
  • Streptomyces / enzymology*
  • Streptomyces / metabolism

Substances

  • Malates
  • Organophosphorus Compounds
  • 2-phosphinomethylmalic acid
  • Citrate (si)-Synthase
  • Malate Synthase
  • 2-phosphinomethylmalic acid synthase
  • Oxo-Acid-Lyases