Binding site for fungal beta-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase.
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.
Tomoda
Hiroshi
H
Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, and The Kitasato Institute, 5-9-1 Shirokane, Minato, Tokyo 108-8641, Japan. tomoda@lisci.kitasato-u.ac.jp
Ohbayashi
Naomi
N
Morikawa
Yuko
Y
Kumagai
Hidetoshi
H
Omura
Satoshi
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006224:Cricetinae; D003553:Cystine / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D010450:Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D005658:Fungi / Q000737:chemistry / Q000378:metabolism*; D006904:Hydroxymethylglutaryl-CoA Synthase; D007783:Lactones / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D016415:Sequence Alignment; D014316:Tritium
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D007783:Lactones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014316:Tritium; C054282:antibiotic 1233A; D003553:Cystine; D006904:Hydroxymethylglutaryl-CoA Synthase; D010450:Endopeptidases; D003066:Coenzyme A Ligases