Structural Insights into the CotB2-Catalyzed Cyclization of Geranylgeranyl Diphosphate to the Diterpene Cyclooctat-9-en-7-ol
Structural Insights into the CotB2-Catalyzed Cyclization of Geranylgeranyl Diphosphate to the Diterpene Cyclooctat-9-en-7-ol
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DOI：
10.1021/acschembio.7b00154
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发表时间：
2017-06-01
影响因子：
4
通讯作者：
Kuzuyama, Tomohisa
中科院分区：
文献类型：
--
作者：
Tomita, Takeo;Kim, Seung-Young;Kuzuyama, Tomohisa
二萜环化酶CotB 2催化香叶基香叶基二磷酸（GGPP）环化为三环环辛-9-烯-7-醇，其特征在于5-8-5-稠环骨架。我们以前提出了一个环化级联涉及一个独特的碳碳键重排结合多个氢化物移位，所有发生在一个单一的活性位点。在这里，我们报告的第一个高分辨率的X-射线晶体结构的CotB 2结合底物类似物geranylgeranyl硫代二磷酸（GGSPP）。在GGSPP结合形式中，GGSPP折叠成独特的S形构象，这可能反映了底物GGPP电离之前的底物结合状态。GGSPP的折叠框架被疏水残基和几个芳族和天冬酰胺残基包围，这些残基通过阳离子-p和偶极电荷相互作用的组合被良好定位以稳定一系列反应性碳阳离子中间体。结合晶体结构和基于诱变的生化测定为CotB 2催化过程中精细控制环形成和立体化学提供了结构基础。
The diterpene cyclase CotB2 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to the tricyclic cyclooctat-9-en-7-ol, which is characterized by a 5-8-5-fused ring skeleton. We have previously proposed a cyclization cascade involving a unique carboncarbon bond rearrangement combined with multiple hydride shifts, all occurring at a single active site. Here, we report the first high-resolution X-ray crystal structure of CotB2 with bound substrate analog geranylgeranyl thiodiphosphate (GGSPP). In the GGSPP-bound form, GGSPP folds into a unique S-shaped conformation that probably reflects the substrate-bound state prior to ionization of the substrate GGPP. The folded framework of GGSPP is surrounded by hydrophobic residues and several aromatic and asparagine residues that are well-positioned to stabilize a series of reactive carbocation intermediates through a combination of cation-p and dipole charge interactions. The combined crystal structures and mutagenesis-based biochemical assays provide a structural basis for exquisite control of ring formation and stereochemistry during CotB2 catalysis.