Mechanism and Scope of Prenylated-flavin-dependent (De)carboxylase Enzymes

Regents Of The University Of Michigan - Ann Arbor, Ann Arbor MI

Investigators

Abstract

With the support of the Chemistry of Life Processes (CLP) Program in the Chemistry Division, Professor E. Neil G. Marsh from the University of Michigan is studying how a newly-discovered class of enzymes add and remove carbon dioxide to unreactive molecules to make them more chemically reactive. This research addresses an urgent need to develop new, environmentally friendly routes to make fuels and chemical feedstocks from renewable sources. Understanding how these enzymes work will aid in developing highly efficient “green” catalysts to replace current energy-intensive industrial processes. Enzymes are biocompatible, biodegradable and non-toxic catalysts. Using enzymes and biorenwable feedstocks in industrial processes helps to replace fossil hydrocarbons for the production of commodity chemicals in ways that are cost-effective, low-energy and sustainable. In synergy with the scientific goals, the project will advance the education, training and professional development of undergraduate and graduate students, and postdoctoral scientists, including those from underserved minority groups, in the interdisciplinary area of chemical biology. The project has two scientific goals. The first is to determine how the novel flavin cofactor, prFMN, facilitates (de)carboxylation of aromatic molecules using phenazine-1-carboxylate decarboxylase as a model enzyme. The experimental approaches include: in situ native protein mass spectrometry to identify unstable covalent intermediates formed between substrates and prFMN (prenylated flavin mononucleotide); UV/visible stopped-flow spectroscopy to identify transiently formed intermediates and determine their rates of formation and breakdown and natural abundance 13-C kinetic isotope effect measurements to determine the order of bond-breaking and bond-forming steps, and to provide information on the nature of the transition state. The second is to survey the diversity of prFMN-dependent decarboxylation reactions represented in the prFMN superfamily, with the objective of identifying novel substrates and new (de)carboxylases. The experimental approach will involve screening potential substrates for carboxylation using compound libraries that contain aromatic, heterocyclic, or conjugated double bond functionalities. Potential substrates will be identified by their ability to undergo H/D exchange when incubated with the appropriate enzyme. Many compounds will be screened in parallel using LC-MS (liquid chromatography with mass spectral detection). The approach will be applied to sample a diverse cross-section of the prFMN superfamily. These studies are expected to provide fundamental information about the mechanisms of this important class of enzymes. 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.