Thousands of terpenoid derivatives found throughout Nature are involved in diverse biosynthetic and metabolic pathways such as cholesterol biosynthesis in humans and menthol biosynthesis in mint. Notably, many terpenoids have been used as medicinal agents since the times of antiquity due to their analgesic, antibiotic, and antifungal properties. In spite of the universal importance of this family of natural products for human health, the three-dimensional structures of terpenoid cyclases have only been reported relatively recently;the majority of these structure determinations have been supported by GM56838. Terpenoid cyclases (a.k.a. terpene synthases) catalyze the cyclization of a common allylic pyrophosphate substrate, such as farnesyl diphosphate, to form one of hundreds of possible products. The terpenoid cyclase plays a critical role as a template in chaperoning substrate and intermediate conformations. The cyclization reaction can be very specific and lead to the formation of one exclusive product, or it can be somewhat promiscuous and lead to the formation of several products. Thus, the terpenoid cyclases comprise an exciting class of biosynthetic enzymes from both the biological and the chemical perspectives. In the current funding period, we have determined the X-ray crystal structures of the sesquiterpene cyclases A. terreus aristolochene synthase, delta-cadinene synthase, and epi-isozizaene synthase;we have established the structural basis for aberrant product formation by wild-type trichodiene synthase and its site-specific mutants;and we have generated the first crystals of a diterpene cyclase, copalyl diphosphate synthase.
We aim to build upon this outstanding structural foundation in the next funding period by dissecting detailed structure-function relationships in aristolochene synthase and epi-isozizaene synthase to better understand the structural basis of biosynthetic diversity. Specifically, we will study site- specific variants engineered to generate alternative products, and we will develop a structure-based approach for generating new cyclic terpenoids in protein engineering experiments. Additionally, we will determine the X-ray crystal structures of copalyl diphosphate synthase and geosmin synthase to explore the evolution of domain architecture in multidomain terpenoid cyclases. These studies will illuminate the evolutionary roots of biosynthetic diversity in the greater family of terpenoid synthases.

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 with great specificity and efficiency will ultimately enable drug discovery at the interface of synthetic chemistry and synthetic biology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function E Study Section (MSFE)
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Smith, Ward
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University of Pennsylvania
Schools of Arts and Sciences
United States
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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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