TY - JOUR
T1 - Binding site for fungal β-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase
AU - Tomoda, Hiroshi
AU - Ohbayashi, Naomi
AU - Morikawa, Yuko
AU - Kumagai, Hidetoshi
AU - Omura, Satoshi
N1 - Funding Information:
This work was supported in part by grants from the “Research for the Future” Program of the Japan Society for the Promotion of Science (JSPS-RFTF96I00304), from the 21st Century COE Program, Ministry of Education, Culture, Sports, Science and Technology, Japan, and from Uehara Memorial Foundation, Japan.
PY - 2004/2/27
Y1 - 2004/2/27
N2 - 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.
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.
KW - Binding site
KW - Cytosolic HMG-CoA synthase
KW - Hymeglusin
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U2 - 10.1016/j.bbalip.2003.11.005
DO - 10.1016/j.bbalip.2003.11.005
M3 - Article
C2 - 14984735
AN - SCOPUS:1242292240
SN - 1388-1981
VL - 1636
SP - 22
EP - 28
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
IS - 1
ER -