Mechanism of a new flavin-derived cofactor involved in enzymatic decarboxylation reactions

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Neil Marsh from the University of Michigan for studies on a potentially useful new enzyme. Enzymes are proteins that speed-up many chemical reactions within cells and thus, enzymes are necessary for life. There is increasing interest in using enzymes to catalyze industrial chemical reactions because they are extremely efficient, renewable, biodegradable and non-toxic. One class of industrially promising enzymes is known as decarboxylases. These enzymes have the ability to add carbon dioxide to (or remove carbon dioxide from) molecules, which may allow them to be used in processes to develop new biofuels or important commodity chemicals such as styrene (used in making plastics). This project is studing the biochemical properties of a newly discovered decarboxylase enzyme with the long-term objective of producing this enzyme for industrial applications. In synergy with the scientific goals, the project is advancing the education, training and professional development of undergraduates, graduate students and postdoctoral scientists in the inter-disciplinary area of chemical biology. Students and postdocs are receiving training in a wide variety of specialized techniques ranging from organic synthesis to sophisticated spectroscopy experiments. The project investigates the mechanism of a novel 2-component decarboxylase system recently identified from yeast. Ferulic acid decarboxylase (FDC) catalyzes the decarboxylation of a wide range of substituted phenylacrylic acids and sorbic acid. The enzyme uses a novel, modified flavin mononucleotide (FMN) as a cofactor that contains an additional 6-membered ring derived from dimethylallyl phosphate. The second enzyme, termed PAD1, synthesizes the cofactor from reduced FMN and dimethylallyl phosphate. In particular, the Marsh group aims to determine the mechanism of FDC and role of the novel, modified flavin cofactor in catalyzing the decarboxylation reaction. They further aim to determine the mechanism by which PAD1 synthesizes the modified flavin cofactor. The experimental approaches includes measuring the kinetics of the FDC- and PAD1-catalyzed reactions using both steady state and pre-steady state techniques with a variety of substrate and coenzyme analogs. Deuterium, and 14C and 13C kinetic isotope effects are measured for FDC and combined with computational studies to fit the experimentally-determined kinetic isotope effects with models of the enzyme transition state.