The proposed research explores the structure and mechanism of terpenoid cyclases, which are unique among enzymes in that they catalyze the most complex carbon-carbon bond forming reactions in biology: 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 terpenoid cyclase structure and mechanism will enable drug discovery and manufacture at the interface of natural products chemistry, enzymology, structural biology, and synthetic biology. To advance our understanding of structure-function relationships in terpenoid cyclases, we will pursue the following lines of investigation: (1) We will determine the structural basis of reprogrammed cyclization cascades catalyzed by site-specific mutants of the C15 sesquiterpene cyclase epi-isozizaene synthase from Streptomyces coelicolor (EIZS). We will study the temperature dependence of cyclization fidelity in selected EIZS mutants and we will determine crystal structures of complexes with analogues of substrate and carbocation intermediates, as well as the C15 hydrocarbon product. These snapshots of wild-type and mutant EIZS cyclization cascades will show us how cyclization chemistry can be redirected to generate alternative products. (2) We will determine the structural basis of cyclization fidelity in the bifunctional C20 diterpene cyclase fusicoccadiene synthase from Phomopsis amygdali (PaFS). We will study the temperature dependence of PaFS cyclization fidelity and we will determine crystal structures of complexes with substrate analogues and the hydrocarbon product to generate snapshots of the diterpene cyclization cascade. We will also study a double mutant designed to introduce C25 sesterterpene cyclase activity by increasing active site volume. These structures will be the first to map out the reaction coordinate of a diterpene cyclization reaction. (3) We will determine the structural basis of assembly-line biosynthesis in the bifunctional C25 sesterterpene synthases ophiobolin F synthase from Aspergillus clavatus (AcOS) and mangicdiene synthase from Fusarium graminearum (FgMS). We will develop a radiolabeled substrate to measure the steady-state kinetics of sesterterpene hydrocarbon formation, and we will determine whether channeling occurs between the prenyltransferase and cyclase active sites of each bifunctional enzyme. We will also determine structures of AcOS and FgMS using X-ray crystallography and/or electron microscopy to better understand assembly-line terpenoid biosynthesis. These studies will provide the first structural views of sesterterpene synthases, a family of terpenoid biosynthetic enzymes discovered only recently.

Public Health Relevance

Structural and functional studies of the terpenoid cyclases show how these novel enzymes generate the largest and most diverse family of natural products found on the Earth. Importantly, many terpenoids exhibit useful medicinal properties, e.g., as antibacterial, antifungal, anti-inflammatory, or anticancer agents. Therefore, understanding and engineering terpenoid cyclase function in generating complex carbon scaffolds will ultimately enable drug discovery at the interface of natural products chemistry, enzymology, structural biology, and synthetic biology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Bond, Michelle Rueffer
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University of Pennsylvania
Schools of Arts and Sciences
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Blank, Patrick N; Pemberton, Travis A; Chow, Jeng-Yeong et al. (2018) Crystal Structure of Cucumene Synthase, a Terpenoid Cyclase That Generates a Linear Triquinane Sesquiterpene. Biochemistry 57:6326-6335
Christianson, David W (2017) Structural and Chemical Biology of Terpenoid Cyclases. Chem Rev 117:11570-11648
Pemberton, Travis A; Chen, Mengbin; Harris, Golda G et al. (2017) Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases. Biochemistry 56:2010-2023
Blank, Patrick N; Barrow, Golda H; Chou, Wayne K W et al. (2017) Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes. Biochemistry 56:5798-5811
Christianson, David W; Scrutton, Nigel S (2016) Editorial overview: Catalysis and regulation: enzyme structure, mechanism, and biosynthetic pathways. Curr Opin Struct Biol 41:viii-x
Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu et al. (2016) Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase. ACS Chem Biol 11:889-99
Chen, Mengbin; Harris, Golda G; Pemberton, Travis A et al. (2016) Multi-domain terpenoid cyclase architecture and prospects for proximity in bifunctional catalysis. Curr Opin Struct Biol 41:27-37
Pemberton, Travis A; Christianson, David W (2016) General base-general acid catalysis by terpenoid cyclases. J Antibiot (Tokyo) 69:486-93
Chen, Mengbin; Chou, Wayne K W; Al-Lami, Naeemah et al. (2016) Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase. Biochemistry 55:2864-74
Grundy, Daniel J; Chen, Mengbin; González, Verónica et al. (2016) Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture. Biochemistry 55:2112-21

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