Structure and Function of Biosynthetic Enzymes

The proposed research explores the structure and mechanism of terpene cyclases, which are unique among enzymes in that they catalyze the most complex carbon-carbon bond forming reactions in nature: on average, more than half of the substrate carbon atoms undergo changes in bonding and/or hybridization during the course of a typical enzyme-catalyzed reaction. Notably, many terpenoids exhibit useful pharmacological properties, such as the blockbuster cancer chemotherapy drug Taxol (paclitaxel) and the antimalarial drug artemisinin. Thus, a better understanding of terpene cyclase structure and mechanism will enable drug discovery and manufacturing at the interface of natural products chemistry, enzymology, structural biology, and synthetic biology. To advance our understanding of structure-function relationships in terpene cyclases, we will pursue the following lines of investigation: (1) We will determine the structural basis of substrate binding, transit, and catalysis in a class I assembly- line terpene synthase, fusicoccadiene synthase from Phomopsis amygdali (PaFS). We will determine cryo-EM structures of PaFS complexes with an inhibitor and with a substrate analogue, and we will determine the influence of oligomeric structure as well as the interdomain linker on substrate channeling between the prenyltransferase and cyclase domains. These studies will broaden our understanding of substrate channeling – perhaps better designated as "directed substrate transit" – between covalently-linked enzymes catalyzing consecutive reactions in a biosynthetic pathway. (2) We will determine the structural basis of substrate binding, transit, and catalysis in class II assembly- line terpene synthases, the copalyl diphosphate synthases from Penicillium verruculosum (PvCPS) and Penicillium fellutanum (PfCPS). We will complete the cryo-EM structure determination of PfCPS, and we will determine the cryo-EM structures of its complexes with a substrate analogue and product. We will also determine whether directed substrate transit occurs between the prenyltransferase and cyclase domains in both PfCPS and PvCPS, and we will ascertain the importance of oligomeric structure for catalytic function. (3) We will explore and exploit the structural basis of chemodiversity in terpene biosynthesis, focusing on sesquiterpene synthases that quench reactive carbocation intermediates with hydroxyl or amino nucleophiles. We will convert our paradigm for protein engineering, epi-isozizaene synthase, into a sesquiterpene alcohol synthase. We will also determine structure-function relationships for the sesquiterpene synthase FlvF from Aspergillus flavus to understand how it catalyzes the condensation of a cyclic sesquiterpene with dimethylcadaverine. Importantly, FlvF represents the first example of a synthase that catalyzes C–N bond formation with a cyclic terpene.