Tomita Takeo, Kim Seung-Young, Teramoto Kazuya, Meguro Ayuko, Ozaki Taro, Yoshida Ayako, Motoyoshi Yudai, Mori Naoki, Ishigami Ken, Watanabe Hidenori, Nishiyama Makoto, Kuzuyama Tomohisa
RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
ACS Chem Biol. 2017 Jun 16;12(6):1621-1628. doi: 10.1021/acschembio.7b00154. Epub 2017 May 2.
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 carbon-carbon 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-π 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.
二萜环化酶CotB2催化香叶基香叶基二磷酸（GGPP）环化生成具有5-8-5稠合环骨架特征的三环环辛-9-烯-7-醇。我们之前提出了一种环化级联反应，该反应涉及独特的碳-碳键重排以及多个氢化物转移，所有这些都发生在单个活性位点。在此，我们报道了首个结合有底物类似物香叶基香叶基硫代二磷酸（GGSPP）的CotB2的高分辨率X射线晶体结构。在结合GGSPP的形式中，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以及几个芳香族和天冬酰胺残基包围，这些残基通过阳离子-π和偶极电荷相互作用的组合，很好地定位以稳定一系列反应性碳正离子中间体。结合的晶体结构和基于诱变的生化分析为CotB2催化过程中环形成和立体化学的精确控制提供了结构基础。
