Proposed mechanism for the reaction catalyzed by a diterpene cyclase, aphidicolan-16beta-ol synthase: experimental results on biomimetic cyclization and examination of the cyclization pathway by ab initio calculations.
To examine the mechanism of the cyclization reaction catalyzed by aphidicolan-16beta-ol synthase (ACS), which is a key enzyme in the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha, skeletal rearrangement of 2a and biomimetic cyclization of 4b were employed. The structures of the reaction products, which reflect penultimate cation intermediates, allowed us to propose a detailed reaction pathway for the Lewis acid-catalyzed cyclizations and rearrangements. Isolation of these products in an aphidicolin-producing fungus led us to speculate that the mechanism of the ACS-catalyzed cyclization reaction is the same as that of a nonenzymatic reaction. Ab initio calculations of the acid-catalyzed reaction intermediates and the transition states indicate that the overall reaction catalyzed by ACS is an exothermic process though the reaction proceeds via an energetically disfavored secondary cation-like transition state. In conjunction with the solvent effect in the acid-catalyzed reactions, this indicates that the actual role of ACS is to provide a template which enforces conformations of the intermediate cations leading to the productive cyclization although it has been believed that the cation-pi interaction between cation intermediates and aromatic amino acid residues in the active site is important for the enzymatic catalysis. This study provided important information on the role of various cationic species, especially secondary cation-like structures, in both nonenzymatic and enzymatic reactions.
Oikawa
Hideaki
H
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. hoik@chem.agr.hokudai.ac.jp
Nakamura
Kensuke
K
Toshima
Hiroaki
H
Toyomasu
Tomonobu
T
Sassa
Takeshi
T
D000164:Acremonium / Q000201:enzymology; D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D002384:Catalysis; D002849:Chromatography, Gas; D019295:Computational Biology; D003500:Cyclization; D004224:Diterpenes / Q000737:chemistry*; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical
D004224:Diterpenes; D007202:Indicators and Reagents; D019883:Alkyl and Aryl Transferases; C433216:aphidicolan-16beta-ol synthase