首页> 美国卫生研究院文献>Wiley-Blackwell Online Open >Nucleophilic Water Capture or Proton Loss: Single Amino Acid Switch Converts δ‐Cadinene Synthase into Germacradien‐4‐ol Synthase
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Nucleophilic Water Capture or Proton Loss: Single Amino Acid Switch Converts δ‐Cadinene Synthase into Germacradien‐4‐ol Synthase

机译:亲核水捕获或质子损失:单个氨基酸开关将δ-卡丁烯合酶转化为Germacradien-4-ol合酶

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摘要

δ‐Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ‐cadinene and about 2 % germacradien‐4‐ol, which is also generated from FDP by the cyclase germacradien‐4‐ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site‐directed mutagenesis of δ‐cadinene synthase. If W279 in δ‐cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. DCS‐W279A is a catalytically highly efficient germacradien‐4‐ol synthase (k cat/K M=1.4×10−3 μm s−1) that produces predominantly germacradien‐4‐ol in addition to 11 % δ‐cadinene. Water capture is not achieved through strategic positioning of a water molecule in the active site, but through a coordinated series of loop movements that allow bulk water access to the final carbocation in the active site prior to product release.
机译:δ-卡丁烯合酶是一种倍半萜烯环化酶,它利用通用的非手性前体法呢基二磷酸二磷酸酯(FDP)产生主要是双环倍半萜烯δ-cadinene和约2%的芽孢ac烯-4-醇,这也是由环化酶芽孢杆菌四烯从FDP产生的。 -ol合酶。本文中,通过δ-cadinene合酶的定向诱变研究了倍半萜烯合酶区分去质子化和与亲核水分子反应的机理。如果用各种较小的氨基酸替换δ-cadinene合酶中的W279,则醇与烃产物的比例与氨基酸侧链的范德华体积成正比。 DCS‐W279A是一种催化高效的胚芽四醇合酶(k cat / KM = 1.4×10 −3 μms −1 ),主要产生胚芽四氢嘧啶-4除了11%δ-卡丹烯外,还提供‐ol。不能通过战略性地将水分子定位在活性位点上来实现集水,而是通过一系列协调的循环运动来实现,这些循环运动允许大量水在产品释放之前进入活性位点中的最终碳正离子化。

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