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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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
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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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