Bifunctional Abietadiene Synthase: Free Diffusive Transfer of the (+)-Copalyl Diphosphate Intermediate between Two Distinct Active Sites

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• 作者：Reuben J. Peters ; Matthew M. Ravn ; Robert M. Coates ; and Rodney B. Croteau
• 刊名：Journal of the American Chemical Society
• 出版年：2001
• 出版时间：September 19, 2001
• 年：2001
• 卷：123
• 期：37
• 页码：8974 - 8978
• 全文大小：65K
• 年卷期：v.123,no.37(September 19, 2001)
• ISSN：1520-5126

文摘

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in theconversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial stepof resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphateto the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the secondreaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-typebackbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristicof the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried outat a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity andthe location of these motifs suggests that the two active sites reside in separate domains. These mutants effectivelycomplement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two activesites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thusconclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhancethe activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implicationsof these results for the kinetic mechanism of abietadiene synthase are discussed.