With this award, the Chemistry of Life Processes Program in the Chemistry Division and the Molecular Biophysics Cluster in the Molecular and Cellular Biosciences Division are funding Dr. Pablo Sobrado from Virginia Tech and Dr. John J. Tanner from the University of Missouri-Columbia to study a new class of flavoenzymes in which the flavin does not play a redox role in catalysis. Enzymes that contain riboflavin (vitamin B2) are known as flavoenzymes. In general, flavoenzymes utilize the properties of the flavins to catalyze oxidation/reduction chemistry. These reactions involve movement of electrons and changes in oxidation state as one compound is converted into another. Recently, new functionalities have been demonstrated for several flavoenzymes that do not involve changes in oxidation state for the flavin or the compounds involved in the reactions. For example, the flavin can act as an acid or base instead. Such flavoenzymes are referred to as atypical. One focus of the project is to discover the protein-flavin interactions that tune the flavin for this novel reactivity. The second objective is the characterization of 2-haloacrylate hydratase (2-HAH), an atypical flavoenzyme involved in bioremediation of halogenated compounds. Preliminary data suggest that the way the 2-HAH reaction is catalyzed may be unprecedented. Graduate and undergraduate students participating in this project will gain interdisciplinary training in chemistry, biochemistry, and structural biology.
The class of atypical flavoenzymes has been much less studied compared to traditional flavin oxidoreductases. The overarching goal of this proposal is to identify the conserved three-dimensional structural features of atypical flavoenzymes that enable the flavin to be a nucleophile, electrophile, or acid/base rather than a traditional redox cofactor. Two atypical flavoenzymes will be studied: 1) UDP-galactopyranose mutase (UGM) and 2) 2-haloacrylate hydratase (2-HAH). The main goal is to formulate the biochemical and structural principles that govern the chemical reactivity of atypical flavoenzymes. The analysis of the available structural and mechanistic data on UGM resulted in a hypothesis about the structural and dynamic elements that endow flavins with new chemical activities. This hypothesis will be tested using site directed mutagenesis of UGM, X-ray crystallography, and molecular dynamics simulations. 2-HAH is a recently identified flavoprotein that catalyzes the formation of pyruvate from 2-haloacrylate. There is no net-redox change in this reaction; however, there is an absolute requirement for the reduced flavin for activity. In addition, light greatly enhances the rate of reduction of the enzyme by NAD(P)H, which is not normally seen for atypical flavoenzymes. The role of the reduced flavin in the 2-HAH reaction and the mechanism of light activation will be investigated. X-ray crystallography will be employed to solve the three-dimensional structure of 2-HAH.
