NAD(P)H: (Quinone acceptor) oxidoreductase (QR) is a FAD-containing flavoprotein that catalyzes obligatory two-electron reductions of quinones to hydroquinones. QR is induced by a variety of xenobiotics in many tissues and cell lines where it is a major system for the detoxification of quinones and their metabolic precursors. The protective effects of many anticarcinogens may be attributed at least in part to their ability to induce QR. The enzyme also catalyzes the reductive activation of mitomycin C and other chemotherapeutic quinones. Thus, the same reaction that protects normal cells from quinones, activates the compounds that lead to tumor cell death. The mechanism by which this unusual flavoprotein carries out two-electron reductions is starting to unravel in large part as a result of the structural and mechanistic data obtained in this project. The long-term goal of this project is to characterize more fully the mechanism of this reaction. QR is also exquisitely sensitive to inhibition by dicoumarol and related anticoagulants, and may play an important role in the biosynthesis of certain blood coagulation proteins by generating reduced vitamin K required for the gamma-carboxylation of glutamate residues. As part of this project, the applicant has prepared diffraction quality crystals of mouse and rat liver QR, determined the three-dimensional structure of four complexes of rat liver QR, proposed a mechanism for the obligatory two-electron reaction, and determined and refined the structure of mouse QR in two crystal forms. In the next period he proposes to further characterize the mechanism of the reaction and study the activation of chemotherapeutic agents by pursuing the following aims: 1) elucidation of the mechanism of the final step in quinone reduction through the determination of the structures of QR with other substrates, substrate analogs, and products; 2) characterization of the mechanism of flavin reduction by studying the structures of QR with nicotinamide and flavin analogs; 3) analysis of the interactions and the activation by QR of mitomycin C and adriamycin - two quinolic therapeutic agents; 4) determination of the structures of the complexes of rat and/or mouse QR with experimental chemotherapeutic agents activated by QR to provide the basis for the design of improved agents; 5) crystal improvement and structure analysis of the recombinant human QR.
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