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
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Koksal, Mustafa; Potter, Kevin; Peters, Reuben J et al. (2014) 1.55A-resolution structure of ent-copalyl diphosphate synthase and exploration of general acid function by site-directed mutagenesis. Biochim Biophys Acta 1840:184-90
Li, Ruiqiong; Chou, Wayne K W; Himmelberger, Julie A et al. (2014) Reprogramming the chemodiversity of terpenoid cyclization by remolding the active site contour of epi-isozizaene synthase. Biochemistry 53:1155-68
Thomas, Jemima C; Matak-Vinkovic, Dijana; Van Molle, Inge et al. (2013) Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases. Biochemistry 52:5236-46
Chen, Mengbin; Al-lami, Naeemah; Janvier, Marine et al. (2013) Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase. Biochemistry 52:5441-53
Koksal, Mustafa; Chou, Wayne K W; Cane, David E et al. (2013) Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase. Biochemistry 52:5247-55
Chen, Mo; Drury, Jason E; Christianson, David W et al. (2012) Conversion of human steroid 5?-reductase (AKR1D1) into 3?-hydroxysteroid dehydrogenase by single point mutation E120H: example of perfect enzyme engineering. J Biol Chem 287:16609-22
Chen, Mo; Adeniji, Adegoke O; Twenter, Barry M et al. (2012) Crystal structures of AKR1C3 containing an N-(aryl)amino-benzoate inhibitor and a bifunctional AKR1C3 inhibitor and androgen receptor antagonist. Therapeutic leads for castrate resistant prostate cancer. Bioorg Med Chem Lett 22:3492-7
Koksal, Mustafa; Chou, Wayne K W; Cane, David E et al. (2012) Structure of geranyl diphosphate C-methyltransferase from Streptomyces coelicolor and implications for the mechanism of isoprenoid modification. Biochemistry 51:3003-10
Koksal, Mustafa; Chou, Wayne K W; Cane, David E et al. (2012) Structure of 2-methylisoborneol synthase from Streptomyces coelicolor and implications for the cyclization of a noncanonical C-methylated monoterpenoid substrate. Biochemistry 51:3011-20
Koksal, Mustafa; Jin, Yinghua; Coates, Robert M et al. (2011) Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis. Nature 469:116-20

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