Reaction mechanism of unique terpene synthases, that is elucidated by synthetic chemistry and structural biology

2016 Fiscal Year Final Research Report

• Project/Area Number: 26292058
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Bioorganic chemistry
• Research Institution: The University of Tokyo
• Principal Investigator: Kuzuyama Tomohisa 東京大学, 生物生産工学研究センター, 准教授 (30280952)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Keywords: 生合成 / 反応機構 / テルペン / 環化酵素

Outline of Final Research Achievements

We identified diterpene cyclase CotB2 by sequence analysis and heterologous expression and demonstrated that CotB2 synthesizes cyclooctat-9-en-7-ol with six chiral centers from the achiral geranylgeranyl diphosphate substrate. We revealed an unusual mechanism for the exquisite CotB2-catalyzed cyclization that involves a carbon－carbon backbone rearrangement and two long-range hydrogen migrations, based on a combination of in vivo and in vitro studies with stable-isotope-labeled substrates and theoretical calculations. We also succeeded in solving the first high-resolution X-ray crystal structure of CotB2 with bound substrate analog geranylgeranyl thiodiphosphate. The combined crystal structures and mutagenesis-based biochemical assays provided structural basis for exquisite control of ring formation and stereochemistry during CotB2 catalysis. In addition, we identified and characterized a unique monoterpene synthase, cyclolavandulyl diphosphate synthase.

Free Research Field

天然物化学