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.
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
Hiroshi
H
Tomoda
Naomi
N
Ohbayashi
Yuko
Y
Morikawa
Hidetoshi
H
Kumagai
Satoshi
S
Omura
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