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

Hiroshi Tomoda, Naomi Ohbayashi, Yuko Morikawa, Hidetoshi Kumagai, Satoshi Omura

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

We studied the molecular mechanism through which the fungal β-lactone, hymeglusin, potently and specifically inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. [14C]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 [ 14C]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 [3H]hymeglusin was digested with V8 protease, and the [3H]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-[3H] 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.

Original languageEnglish (US)
Pages (from-to)22-28
Number of pages7
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1636
Issue number1
DOIs
StatePublished - Feb 27 2004

Fingerprint

Hydroxymethylglutaryl-CoA Synthase
Lactones
Coenzyme A
Binding Sites
Cricetinae
fluorophosphate
Enzymes
Iodoacetamide
Peptides
Ethylmaleimide
Catalytic Domain
High Pressure Liquid Chromatography
Liver

Keywords

  • Binding site
  • Cytosolic HMG-CoA synthase
  • Hymeglusin

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Biophysics

Cite this

Binding site for fungal β-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase. / Tomoda, Hiroshi; Ohbayashi, Naomi; Morikawa, Yuko; Kumagai, Hidetoshi; Omura, Satoshi.

In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, Vol. 1636, No. 1, 27.02.2004, p. 22-28.

Research output: Contribution to journalArticle

Tomoda, Hiroshi ; Ohbayashi, Naomi ; Morikawa, Yuko ; Kumagai, Hidetoshi ; Omura, Satoshi. / Binding site for fungal β-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase. In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 2004 ; Vol. 1636, No. 1. pp. 22-28.
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AB - We studied the molecular mechanism through which the fungal β-lactone, hymeglusin, potently and specifically inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. [14C]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 [ 14C]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 [3H]hymeglusin was digested with V8 protease, and the [3H]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-[3H] 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.

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