Exploring the Mechanistic Diversity of Prenylated-Flavin-Dependent Enzymes

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There is a need to develop new, environmentally friendly routes to fuels and chemical feedstocks from renewable sources. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Neil Marsh from the University of Michigan Ann Arbor to examine how a newly-discovered class of enzymes remove carbon dioxide from unreactive starting materials to make them more chemically reactive and likely to form value-added commodity chemicals. Understanding the basic mechanisms of reaction for these enzymes will allow the development of highly efficient "green" catalysts to potentially replace energy-intensive industrial processes. Such biocompatible, biodegradable and non-toxic catalysts dovetail with efforts to replace fossil hydrocarbons for the production of commodity chemicals that are cost-effective, low-energy and sustainable. In synergy with the scientific goals, the project advances the education, training and professional development of undergraduate and graduate students, and postdoctoral scientists (including those from underrepresented minority groups) in the interdisciplinary area of chemical biology.The project will elucidate the mechanisms of three prenylated-flavin (prFMN) decarboxylases that appear to use the same cofactor to catalyze decarboxylation reactions through three distinct mechanisms. These enzymes are: ferulic acid decarboxylase in which prFMN is hypothesized to react by a dipolar cycloaddition mechanism; 4-hydroxybenzoate decarboxylase in which prFMN may react by an electrophilic mechanism; and phenazine-1-carboxylate decarboxylase for which evidence suggests prFMN may react by a radical mechanism. The experimental approaches include steady state and pre-steady state kinetic analyses combined with isotopically-labelled substrates. These studies determine the rates of formation and breakdown of enzyme intermediates. The researches also identify unstable covalent intermediates formed between substrates and prFMN using novel mass spectrometry methods that allow for the analysis of proteins in their native state. Finally, the researchers will probe the reaction pathways with substrate analogs that may function as mechanism-based inhibitors.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.