Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases

Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated as alpha, beta and gamma. Catalysis by class I terpenoid synthases occurs exclusively in the a domain, which is found with alpha, alpha alpha, alpha beta gamma, and alpha beta gamma domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, alpha beta gamma), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (alpha alpha)(6)), and ophiobolin F synthase from Aspergillus clavatus (AcOS, alpha alpha). We show that the cyclization fidelity and catalytic efficiency of the alpha domain of TbTS are severely compromised by deletion of the beta gamma domains; however, retention of the beta domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one a domain similarly influences catalysis by the other alpha domain [Chen, M., Chou, W. K. W., Toyomasu, T., Cane, D. E., and Christianson, D. W. (2016) ACS Chem. Biol. 11, 889-899]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the alpha domains of PaFS and AcOS can be swapped so as to make functional chimeric alpha alpha synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C-20 diterpene products and three C-25 sesterterpene products are generated by these chimeras. Thus, engineered alpha beta gamma and alpha alpha terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.