Cyclic terpenoids are found throughout nature but comprise an especially important class of compounds from plants which serve as pharmaceutical agents with antibiotic, antifungal and antitumor activities. The biosynthesis of cyclic terpenes is determined by key branch point enzymes referred to as terpene cyclases, or more properly, terpene synthases. The objective of this proposal is to understand as completely as possible the structural, functional, and chemical features governing the cyclizations of farnesyl diphosphage (FPP) catalyzed by highly homologous plant sesquiterpene cyclases and one diterpene cyclase. The proposed work relies on our recent success with a domain-swapping strategy to map and substitute functional domains between sesquiterpene cyclase genes, and enzymological characterization of the bacterial expressed cyclase proteins. We are now proposing to use new crystallographic data of the wildtype and mutant enzymes in an iterative process with domain swapping and site directed mutagenesis to identify, in much greater detail, those domains and amino acid residues essential for catalysis of particular partial steps within the cyclase reactions. The proposed experimental plan will provide the foundation for our long range goal that focuses on a rationally based redesign of terpene cyclases for the enzymatically directed synthesis of pharmaceutically important terpenoids or their synthetic precursors. This structure/function analysis should also increase our fundamental appreciation for terpene enzymology and biosynthesis in general, and the biosynthesis of cyclic terpenoids like cholesterol, steroid hormones, bile acids, carotenoids and retinoids, and lipid soluble vitamins, A,D,E, and K in particular.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054029-02
Application #
2392276
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1996-04-01
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
005436803
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Rising, Kathleen A; Crenshaw, Charisse M; Koo, Hyun Jo et al. (2015) Formation of a Novel Macrocyclic Alkaloid from the Unnatural Farnesyl Diphosphate Analogue Anilinogeranyl Diphosphate by 5-Epi-Aristolochene Synthase. ACS Chem Biol 10:1729-36
Faraldos, Juan A; Wu, Shuiqin; Chappell, Joe et al. (2010) Doubly deuterium-labeled patchouli alcohol from cyclization of singly labeled [2-(2)H(1)]farnesyl diphosphate catalyzed by recombinant patchoulol synthase. J Am Chem Soc 132:2998-3008
O'Maille, Paul E; Malone, Arthur; Dellas, Nikki et al. (2008) Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases. Nat Chem Biol 4:617-23
Takahashi, Shunji; Yeo, Yunsoo; Greenhagen, Bryan T et al. (2007) Metabolic engineering of sesquiterpene metabolism in yeast. Biotechnol Bioeng 97:170-81
Takahashi, Shunji; Yeo, Yun-Soo; Zhao, Yuxin et al. (2007) Functional characterization of premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and stereo-specific hydroxylations of diverse sesquiterpene substrates. J Biol Chem 282:31744-54
Faraldos, Juan A; Zhao, Yuxin; O'Maille, Paul E et al. (2007) Interception of the enzymatic conversion of farnesyl diphosphate to 5-epi-aristolochene by using a fluoro substrate analogue: 1-fluorogermacrene A from (2E,6Z)-6-fluorofarnesyl diphosphate. Chembiochem 8:1826-33
Greenhagen, Bryan T; O'Maille, Paul E; Noel, Joseph P et al. (2006) Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases. Proc Natl Acad Sci U S A 103:9826-31
O'Maille, Paul E; Chappell, Joe; Noel, Joseph P (2006) Biosynthetic potential of sesquiterpene synthases: alternative products of tobacco 5-epi-aristolochene synthase. Arch Biochem Biophys 448:73-82
Takahashi, Shunji; Zhao, Yuxin; O'Maille, Paul E et al. (2005) Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes. J Biol Chem 280:3686-96
O'Maille, Paul E; Tsai, Ming-Daw; Greenhagen, Bryan T et al. (2004) Gene library synthesis by structure-based combinatorial protein engineering. Methods Enzymol 388:75-91

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