| Budget Amount *help |
¥16,380,000 (Direct Cost: ¥12,600,000、Indirect Cost: ¥3,780,000)
Fiscal Year 2016: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000)
Fiscal Year 2015: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2014: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
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| 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.
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