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Mass Spectrometry Profiling of Terpene Cyclases: Towards Organic Synthesis through Protein Engineering

presented by

Paul E. O’Maille
The Salk Institute for Biological Studies

August 26, 2004

The Scripps Research Institute, W.M. Keck Foundation Amphitheater


Background:

Dr. O’Maille has been involved in research since 1993, when he began with inorganic synthesis as an undergraduate in the laboratory of Prof. Paul Challen at John Carroll University. He later moved on to The Ohio State University, was awarded an NIH predoctoral fellowship, and earned a Ph. D. in Biochemistry from the laboratory of Prof. Ming-Daw Tsai in 2001. His graduate work culminated in the development of structure-based combinatorial protein engineering (SCOPE), which was featured as the cover illustration for the volume of JMB that the resulting publication appeared in. The following year he joined the laboratory of Prof. Joseph P. Noel at The Salk Institute for Biological Studies, where he currently resides. Ongoing research involves X-ray crystallography, protein engineering, and development of analytical techniques for GC-MS analysis of terpenoids conducted under the support of an NIH postdoctoral fellowship. Several manuscripts are currently in press, under review, or in preparation with one patent pending.

Abstract:

Terpenoids constitute the most prevalent and diverse family of natural products known, mediating myriad processes ranging from pathogen defense to pollinator attraction, which can translate to applications such as anti-cancer drugs, pesticides, flavorings, and fragrances. The sesquiterpene cyclase family of enzymes command exquisite stereochemical control over electrophilic transformations to create multi-cyclic, multi-chiral hydrocarbon terpenoid products. Understanding how this family of enzymes converts a common acyclic 15-carbon precursor, farnesyl diphosphate (FPP), into more than 300 distinct hydrocarbon skeletons is of considerable academic and commercial interest. Recently, a comparative analysis between two closely related cyclases has yielded new insights; tobacco 5-epi-aristolochene synthase (TEAS) and Hyoscyamus muticus premnaspirodiene synthase (HPS) share a common reaction mechanism but diverge at the penultimate step of catalysis where either a methyl migration or an alkyl shift occurs to produce 5-epi-aristolochene or premnaspirodiene, respectively. Based on the crystal structure of TEAS and sequence comparison with HPS, both enzymes share an identical active site. TEAS was successfully converted into an HPS enzyme however, by nine mutations that line the periphery of the active site. Because these mutations were made sequentially, the question remains, are all nine mutations required for product selectivity? What other combinations, if any, of these nine mutations are sufficient for this property change? Structure-based combinatorial protein engineering (SCOPE) has enabled the facile synthesis of all combinations of nine mutations (2n combinations = 512 mutants, where n is the number of mutations) and the development of a high throughput GC-MS activity assay has provided the means to functionally characterize these mutants towards answering some of these questions. The fundamental aspects of product specificity control in TEAS and HPS revealed by these investigations have numerous implications for the design of structurally guided mutant libraries of terpene cyclases and the combination of SCOPE and GC-MS greatly accelerates the pace by which they are created and characterized for the identification of novel biosynthetic enzymes.

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