Binding site for fungal beta-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase

Biochim Biophys Acta. 2004 Feb 27;1636(1):22-8. doi: 10.1016/j.bbalip.2003.11.005.

Abstract

We studied the molecular mechanism through which the fungal beta-lactone, hymeglusin, potently and specifically inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. [(14)C]Hymeglusin covalently bound to purified rat liver and to recombinant hamster cytosolic HMG-CoA synthases. The enzyme activity was completely inhibited at a binding ratio of 1.6-2.0 mol [(14)C]hymeglusin/mol HMG-CoA synthase. Incubating the enzyme with 2 mM iodoacetamide (IAA) or 2 mM N-ethylmaleimide (NEM) but not with 1.0 mM diisopropyl fluorophosphates (DFP) completely inhibited the binding, suggesting that hymeglusin binds to a Cys residue of HMG-CoA synthase. Recombinant hamster HMG-CoA synthase labeled with [(3)H]hymeglusin was digested with V8 protease, and the [(3)H]peptide was purified by high performance liquid chromatography (HPLC). The sequence of the peptide was Ser-Gly-Asn-Thr-Asp-Ile-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-[(3)H]hymeglusyl Cys-Tyr-Gly-Gly-Thr-Ala-Ala-Val-Phe-Asn-Ala-Val-Asn-, which corresponds to the active site sequence (from Ser 115 to Asn 141) of hamster HMG-CoA synthase. These findings showed that hymeglusin inhibits hamster cytosolic HMG-CoA synthase by covalently modifying the active Cys 129 residue of the enzyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Carbon Radioisotopes
  • Coenzyme A Ligases / antagonists & inhibitors*
  • Coenzyme A Ligases / chemistry
  • Cricetinae
  • Cystine / chemistry
  • Cytosol / enzymology
  • Endopeptidases
  • Enzyme Inhibitors / pharmacology
  • Fatty Acids, Unsaturated / isolation & purification
  • Fatty Acids, Unsaturated / metabolism*
  • Fatty Acids, Unsaturated / pharmacology
  • Fungi / chemistry
  • Fungi / metabolism*
  • Hydroxymethylglutaryl-CoA Synthase
  • Lactones / isolation & purification
  • Lactones / metabolism*
  • Lactones / pharmacology
  • Liver / enzymology
  • Liver / metabolism
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Rats
  • Recombinant Proteins / antagonists & inhibitors
  • Sequence Alignment
  • Tritium

Substances

  • Carbon Radioisotopes
  • Enzyme Inhibitors
  • Fatty Acids, Unsaturated
  • Lactones
  • Peptide Fragments
  • Recombinant Proteins
  • Tritium
  • antibiotic 1233A
  • Cystine
  • Hydroxymethylglutaryl-CoA Synthase
  • Endopeptidases
  • Coenzyme A Ligases