104(P56) コレステロール生合成律速酵素スクアレンエポキシダーゼの構造と新規酵素阻害剤(ポスター発表の部) [in Japanese] 104(P56) Structure, Function, and Novel Enzyme Inhibitors of Squalene Epoxidase, the Rate-limiting Enzyme of Cholesterol Biosynthesis [in Japanese]
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Squalene epoxidase (SE), one of the rate-limiting enzymes of cholesterol biosynthesis, is a non-P450 flavoprotein monooxygenase that catalyze the conversion of squalene to (3S)2,3-oxidosqualene. Recently cloned human SE cDNA encoded 64kDa membrane bound protein with 574 amino acids, whose deduced amino acid sequence showed 84% and 29 % identity to that of rat and yeast SE, respectively. A truncated recombinant human SE without putative N-terminal transmembrane domain was functionally expressed in E. coli using the pET or the IMPACT T7 system. As in the case of other flavoproteins, SE contained highly conserved FAD-binding motifs; GXGXXG, DG and GD motif. Alanine scanning site-directed mutagenesis experiments demonstrated that the conserved G132, G135, E152, D154, and R154 in the GXGXXG motif (β-sheet-α-helix-β-sheet) are essential for the enzyme activity. Naturally occurring galloyl esters such as (-)-epigallocatechin-3-O-gallate (EGCG) (IC_<50>=0.69μM, K_1=0.74μM), the major components of green tea polyphenols, were found to be novel and potent inhibitors of SE. Synthetic alkyl gallates also showed potent inhibition. In particular, n-dodecyl gallate (DG) (IC_<50>=0.061μM, K_1= 0.033μM) showed highest activity. Kinetic analyses revealed that EGCG was a non-competitive inhibitor of SE, while substrate-like DG inhibited the enzyme in competitive manner. The flavoprotein monooxygenase reaction is thought to proceed through formation of active oxygen species. We hypothesized that the inhibition may be attributed to not only the specific binding of the gallate to the active site of the enzyme but also the radical, active oxygen scavenging activities of the galloyl group. Indeed, when molecular modeling studies were carried out using the crystal structure of bacterial p-hydroxybenzoic acid hydroxylase, one of the most well characterized flavoenzymes and also suffered effective enzyme inhibition by EGCG and DG, it was suggested that EGCG could possibly bind to close proximity of the FAD binding site of the enzyme. Detailed structure activity relationship studies for the galloyl esters are now in progress.
天然有機化合物討論会講演要旨集 (41), 619-624, 1999-09-01
Symposium on the chemistry of natural products