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Jennifer Gehret McCarthy, Ph.D.

Mentor: Janet Smith

jgehret@umich.edu

Thesis Defense: October 15, 2012
"Production of Terminal Alkenes in Natural Product Biosynthesis: Structural Studies of Sulfotransferase and Thioesterase Didomains"

Thesis Abstract:

This thesis investigates a generalized biosynthetic scheme to produce hydrocarbons and natural products with a terminal alkene. Tandem sulfotransferase (ST) and thioesterase (TE) domains act to sulfonate a β-hydroxy group (ST) and to hydrolyze, decarboxylate, and eliminate sulfate (TE) from an acyl carrier protein (ACP)-linked substrate to produce a terminal alkene instead of the carboxylic acid or macrolactone expected from a TE domain. Tandem ST-TE domains occur in the gene cluster for biosynthesis of the natural product curacin A (CurM) and in seven other bacterial genomes. Five of these convert free fatty acids to terminal-alkene hydrocarbons by an olefin synthase (OLS). To understand this novel biosynthetic strategy, the ST and TE domains from CurM (from Moorea producens) and OLS (from Synechococcus sp. PCC 7002) were investigated. Structural and biochemical investigations showed how the CurM and OLS ST and TE domains vary from canonical ST and TE folds to perform chemistry unique to the terminal-alkene producing systems. The CurM and OLS enzymes are similar to each other in structure and activity, but also exhibit subtle differences in substrate specificity, structure, and catalytic efficiency. The tandem ST-TE system is a promising chemical tool for the generation of new molecules or in the production of hydrocarbons for biofuel.
This thesis also includes structural and biochemical characterization of DmmA, an orphan haloalkane dehalogenase (HLD), originally annotated as CurN in the curacin pathway. DmmA has a larger active site than other studied HLDs and is a promising candidate for industrial applications for HLDs that act on larger substrates.

Undergraduate Institution: University of Maryland


Research Profile

Polyketides are secondary metabolites made by bacteria, plants, and fungi. Various members of this group of diverse natural products have been used as antibiotics, anticancer drugs, immunosuppressants and anti-cholesterol agents. The pathways that synthesize these compounds are studied to better understand polyketide production as well as discovering new enzymatic functions to generate various chemical moieties. The hope is to better understand and modify these pathways to produce an array of novel drugs.

I am studying the polyketide synthesis pathway that produces Curacin A, which has been found to have anti-mitotic activity. Of particular interest to me is the final cleavage and terminal double bond formation of the pathway, which contains a previously uncharacterized combination of sulfotransferase and thioesterase domains used to complete the reaction. Knowledge of the three dimensional structure of these domains is integral to understanding how this step functions. I am currently working on obtaining the structures of these domains by x-ray crystallography.

Gehret Figure

Publications

Akey, D.L., Gehret J.J., Kare D., Smith, J.L., Insights from the sea: Structural biology of marine polyketide synthases. Nat Prod Rep. 2012.

Gehret, J.J., Gu, L., Geders, T.W., Brown, W.C., Gerwick, L., Gerwick, W.H., Sherman, D.H., Smith, J.L., Structure and activity of DmmA, a marine haloalkane dehalogenase. Protein Sci. 2012. 21(2): p.239-248.

Gehret, J.J., Gu, L., Gerwick, W.H., Wipf, P., Sherman, D.H., Smith, J.L., Terminal alkene formation by the thioesterase of curacin a biosynthesis: structure of a decarboxylating thioesterase. J Biol Chem, 2011. 286(16): p. 14445-54

Gu L, Wang B, Kulkarni A, Gehret JJ, Lloyd KR, Gerwick L, Gerwick WH, Wipf P, Hakansson K, Smith JL, Sherman DH. Polyketide Decarboxylative Chain Termination Preceded by O-Sulfonation in Curacin A Biosynthesis. J Am Chem Soc. 2009. 131 (44):p. 16033-5

Presentations

Gordon Research Seminar and Conference, Molecular Basis of Microbial One-Carbon Metabolism, August 4-10, 2012 (Bates College, Lewiston, ME). Talk and Poster, "Terminal Alkene Synthesis through Decarboxylation: Biosynthetic Route to Hydrocarbons".

25th Anniversary Symposium of the Protein Society, July 23-27, 2011 (Boston, MA) "Young Investigator Talk" and Poster, "Terminal Alkene Synthesis in Natural Products: Biosynthetic Route to Hydrocarbons"

Ohio Valley Crystallography Symposium, November, 2010 (Toledo, OH) Poster, “Sulfotransferase and thioesterase - working together to produce the terminal olefin in curacin a”

American Crystallographer Association (ACA) Annual Meeting, July 2010 (Chicago, IL) Poster, "A sulfotransferase and thioesterase working together to produce the terminal alkene in curacine A"

American Crystallographer Association (ACA) Annual Meeting, July 2009 (Toronto, Canada) Oral Presentation, "Structural Insights into Terminal Double Bond Formation in the Curacin A Biosynthetic Pathway"

Awards

2012-2013 - EBS Endowment for the Development of Graduate Education (EDGE) Award
2012 - Rackham Travel Grant
2011 - Protein Science Young Investigator Travel Grant/The Protein Society Finn Wold Travel Award
2011 - Rackham Travel Grant
2010 - American Crystallographic Association Travel Grant
2010 - Rackham Travel Grant
2010 - Adam and Mary J. Christman Award (Outstanding Third-Year Student)
2009 - Halvor and Mary Christensen Award (Outstanding Second-Year Student)
2009 - Rackham Travel Grant