Nature has evolved a variety of strategies to cleave unactivated carbon-hydrogen bonds in order to functionalize inert carbon centers. These reactions invariably employ metallocofactors to create powerful oxidants that can abstract hydrogen atoms from the carbon centers to be functionalized, generating carbon-centered radicals. In all well-established enzyme mechanisms involving H abstraction, the cleaved C-H bonds are sp3-hybridized, and the associated homolytic bond-dissociation energies (BDEs) are less than (or equal to) the 104 kcal/mol characteristic of methane. Recently, a growing number of reactions have been identified that imply functionalization of carbon centers that are sp2-hybridized with cleavage of bonds that have BDEs of greater than 105 kcal/mol. This project aims to characterize three such enzymatic reactions involving methylation, methylthiolation, and hydroxylation of sp2-hybridized carbon centers. The first two reactions are catalyzed by enzymes belonging to the radical-SAM superfamily, while the third is catalyzed by an enzyme in the Fe(II)- and ?-ketoglutarate-dependent oxygenase family. The results of these studies should significantly expand our understanding of Nature's strategies for functionalization of unactivated sp2-hybridized carbon centers and reveal details of reaction mechanisms that may prove practically useful in design of therapeutic inhibitors of these processes. )

Public Health Relevance

Reactions in which sp2-hybridized carbon centers are functionalized constitute key steps in the biosynthesis of numerous antibiotic natural products and the maturation of certain tRNAs. The inability to functionalize an sp2-hybridized carbon center in a tRNA that specifies lysine incorporation into proteins is a major risk factor for type I diabetes across all ethnic groups. An understanding of these reactions could suggest strategies for design of new antibiotics and other drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM103268-03
Application #
8686884
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Anderson, Vernon
Project Start
2012-07-02
Project End
2016-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
Blaszczyk, Anthony J; Wang, Roy X; Booker, Squire J (2017) TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. Methods Enzymol 595:303-329
Blaszczyk, Anthony J; Wang, Bo; Silakov, Alexey et al. (2017) Efficient methylation of C2 in l-tryptophan by the cobalamin-dependent radical S-adenosylmethionine methylase TsrM requires an unmodified N1 amine. J Biol Chem 292:15456-15467
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Landgraf, Bradley J; Booker, Squire J (2016) Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase. J Am Chem Soc 138:2889-92
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Maiocco, Stephanie J; Arcinas, Arthur J; Landgraf, Bradley J et al. (2016) Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry. Biochemistry 55:5531-5536
Landgraf, Bradley J; McCarthy, Erin L; Booker, Squire J (2016) Radical S-Adenosylmethionine Enzymes in Human Health and Disease. Annu Rev Biochem 85:485-514
Blaszczyk, Anthony J; Silakov, Alexey; Zhang, Bo et al. (2016) Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase. J Am Chem Soc 138:3416-26
Block, Eric; Booker, Squire J; Flores-Penalba, Sonia et al. (2016) Trifluoroselenomethionine: A New Unnatural Amino Acid. Chembiochem 17:1738-51

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