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, two-thirds of the substrate carbon atoms undergo change in bonding and/or hybridization during the course of a typical cyclization cascade to generate one or more products containing multiple rings and stereocenters. Given the vast chemodiversity of terpenoid natural products, it is notable that many terpenoids exhibit useful pharmacological properties. For example, the taxane diterpene paclitaxel (Taxol) is a blockbuster cancer chemotherapeutic drug, the sesquiterpene artemisinin is an antimalarial drug, and the diterpene ingenol (Picato) is used to treat precancerous actinic keratosis. Thus, understanding terpenoid cyclase function in generating complex carbon scaffolds enables drug discovery at the interface of natural products chemistry, enzymology, structural biology, and synthetic biology. To advance our understanding of structure-function relationships in terpenoid cyclases, and to facilitate innovative approaches for the generation of biologically active terpenoids, we will pursue the following lines of investigation: (1) we will determine how alpha-beta-gamma domain architecture influences catalysis by a diterpene cyclase, using taxadiene synthase as our paradigm. This enzyme catalyzes the first committed step of Taxol biosynthesis in the Pacific yew and has been utilized in synthetic biology approaches. (2) We will determine X-ray crystal structures of epi-isozizaene synthase and its site-specific mutants that generate alternative cyclization products to decipher the three-dimensional code that directs the sesquiterpene cyclization cascade. Ultimately, these studies will allow us to engineer enzymes that generate novel cyclic terpenoid products by design. (3) We will determine the structural and chemical basis for water management strategies in the active sites of terpenoid cyclases that utilize a reactive water molecule to quench the cyclization cascade, namely, germacradien-4-ol synthase and methylisoborneol synthase. We will also learn how the active site of aristolochene synthase, which contains an unreactive water molecule, ensures that this water molecule remains an """"""""innocent bystander"""""""" in catalysis.

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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Smith, Ward
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University of Pennsylvania
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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
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
Grundy, Daniel J; Chen, Mengbin; González, Verónica et al. (2016) Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture. Biochemistry 55:2112-21
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
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
Pemberton, Travis A; Christianson, David W (2016) General base-general acid catalysis by terpenoid cyclases. J Antibiot (Tokyo) 69:486-93
Harris, Golda G; Lombardi, Patrick M; Pemberton, Travis A et al. (2015) Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization-Fragmentation Reaction Sequence. Biochemistry 54:7142-55
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