Flavoenzyme Mechanisms: Redox and Non-redox Reactions

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DESCRIPTION (provided by applicant): The proposed research involves a comprehensive investigation of the relationship of structure to function in monomeric sarcosine oxidase (MSOX), a prototypical member of a superfamily of amine-oxidizing enzymes that contain covalently bound derivatives of the vitamin riboflavin. MSOX is an important catabolic enzyme commonly found in soil bacteria and widely used in the clinical evaluation of renal function. The MSOX superfamily contains several biomedically significant human enzymes, including sarcosine dehydrogenase, an enzyme defective in sarcosinemic patients and monoamine oxidase, a drug target in the treatment of Parkinson's disease and depression. The overall goal of these studies is to gain a deeper understanding of the mechanism of flavoenzyme catalysis, the molecular basis for enzyme specificity and the modulation of the inherent chemical reactivity of flavins by the protein environment. We will determine the crystal structure for various MSOX-substrate complexes in studies that build on our success in obtaining a preliminary 2.0 A resolution structure for the E-S complex with L-proline. Amine oxidation generally requires an unprotonated amino group but the substrates for MSOX are amino acids that exist in solution at neutral pH as unreactive zwitterions. We will evaluate the proposal that substrates are activated for oxidation by MSOX via a mechanism that involves a substantial decrease in the pKa of the enzyme-bound versus the free amino acid. The mechanism of substrate oxidation will be investigated in studies involving a mechanism-based inhibitor, alternate substrates, deuterium isotope effects, stereochemical analysis, mutagenesis and molecular dynamic simulations. We will probe the role of the covalent flavin linkage in reconstitution and structural studies with a MSOX apoprotein preparation containing a mutation that allows noncovalent flavin binding but blocks covalent attachment. The mechanism of covalent flavin attachment will be characterized in in vitro flavinylation studies with wild type apoenzyme or appropriate mutants. These studies represent a multidisciplinary approach, involving a combination of biochemical, structural and modeling efforts.