Mechanism of action and inhibition of dehydrosqualene synthase
Mechanism of action and inhibition of dehydrosqualene synthase
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DOI：
10.1073/pnas.1010907107
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发表时间：
2010-12-14
影响因子：
11.1
通讯作者：
Oldfield, Eric
中科院分区：
文献类型：
--
作者：
Lin, Fu-Yang;Liu, Chia-I;Oldfield, Eric
“头对头”的萜烯合成酶催化类固醇和类胡萝卜素生物合成的第一步：两个类异戊二烯二磷酸酯缩合形成环丙基卡宾二磷酸酯，然后开环。在这里，我们报道了金黄色葡萄球菌脱氢角鲨烯合成酶(CRTM)与其反应中间体前奎烯二磷酸(PSPP)、脱氢角鲨烯(DHS)产物以及一系列抑制剂的络合结构。结果表明，在最初的二磷酸损失时，这样形成的初级碳正离子向蛋白质内部弯曲，与戊烯受体上的C2，3双键反应生成PSPP，两个PSPP链的下三分之二占据的位置与底物中的两个法尼基基本相同。然后，PSPP二磷酸返回到Mg2+团簇进行电离，从而引发下半部反应，生成的DHS被捕获在表面口袋中。这一机制得到以下观察的支持：阳离子抑制剂(有意义的抗感染药物)与其正电荷与环丙基卡宾基团位于同一区域；S-硫代二磷酸盐仅当位于烯丙基位置时才抑制；对11个突变体的活性结果表明两个DXXXD保守结构域都是PSPP电离所必需的；头到尾的类异戊二烯合成酶以及萜类环酶具有与CRTM中发现的电离和烯烃供体位点在空间上重叠的电离和烯烃供体位点。
"Head-to-head" terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg2+ cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.