Flavins as Carbon Transfer Agents in Enzymology

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Flavins are derivatives of vitamin b2 that have long been known to help enzymes move electrons in biological reactions. Interestingly, it is now becoming apparent that flavins can sometimes serve as carbon-carriers. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Bruce Palfey, University of Michigan, to investigate this newly recognized and not well understood type of reaction of flavins. This work is focusing on the way the protein thymidylate synthase controls the transfer and modification of a single carbon atom in an important biochemical reaction that is needed for DNA synthesis. A large number of bacteria use this type of reaction in the pathway to synthesize their DNA, so this project is elucidating a fundamental chemical process that is widespread but poorly understood. The project draws on a variety of chemical methods, some involving advanced technologies that allow very fast processes to be observed. The participants in the project, such as undergraduates and postdoctoral fellows, are receiving training in a unique blend of interdisciplinary scientific techniques, strengthening the capabilities of our technical workforce.Flavin enzyme-chemistry is now extending well beyond the canonical borders set by reduction and oxidation using the transfer of hydride equivalents, thiol/disulfide interchanges, single-electron transfers, and reactions with O2. Among the surprising new findings are enzymes that make iminium adducts with reduced flavins. These iminium adducts undergo a variety of fates depending on the particular enzyme. A flavin-dependent thymidylate synthase (thyX) forms thymine using a methylene derived from methylenetetrahydrofolate (CH2THF) and reducing equivalents derived from pyridine nucleotide. Recent evidence implicates the reduced flavin-iminium adduct as the carbon-transfer agent. Key reactive intermediates are being trapped and their chemical structures are being determined by NMR spectroscopy. Synthetic flavins are being used to learn about key transition states from linear free energy relationships and to gain insight into factors that favor carbon-transfer over possibly competitive hydride transfer. Proton transfers, which must be critical in controlling the transfer of electrophilic carbon, are being studied in detail by transient kinetics in the carbon-transfer route leading to thymidylate. Taken as a whole, this project is defining the catalytic strategies and challenges that many enzymes are now recognized to face.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.