The Chemistry of Dihydropyrimidine Dehydrogenase

点击翻译按钮获取中文摘要

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Graham Moran from Loyola University of Chicago is studying the chemistry of the enzyme dihydropyrimidine dehydrogenase (DPD). The movement of electrons and hydrogen ions in biological processes is fundamental to the chemistry of life. The mechanism of transfer of electrons in biological systems involves complex networks of electron carriers and hydrogen ion donors and remains poorly understood. DPD transfers two electrons from an electron donor to reduce a base, which initiates degradation in a class of bases that includes DNA and RNA. DPD has a complex set of cofactors that transmit the electrons one at a time over long distances, which is unlike any other similar enzyme. The study of this enzyme is expected to advance the understanding of how charge transfer is used to favor specific reactions. The graduate and undergraduate students engaged in this research will gain knowledge and experience in spectrophotometry, transient state kinetics, and X-ray crystallography. Students will also learn in a yearly free virtual kinetics workshop that will illustrate how kinetic ideas are employed to glean information about the properties of molecules.DPD catalyzes the rate limiting step in the degradation of pyrimidine bases but it is significantly different from other dehydrogenases that accomplish similar chemistry. Specifically, DPD contains two flavin cofactors, FAD and FMN, separated by ~56 angstroms and connected by a molecular wire of four iron sulfur centers. The research plan is designed to test the hypotheses that pyrimidine reduction by DPD is the instigating step in the catalysis of base degradation and that NADPH is oxidized to backfill the enzyme and re-establish its two-electron reduced active state; in other words, the oxidative half reaction precedes the reductive half reaction. The research involves the modification of specific charged residues clustered around the FAD and FMN cofactors to slow or stall the passage of electrons through these junctions and thus to reveal different parts of the catalytic cycle. Specifically, mutations that reduce the number of negative charged residues proximal to the FAD will be used to raise the reduction potential of FAD and slow electron transfer to FMN. Similarly, mutations that reduce the number of positively charged residues near the FMN will be used to decrease its capacity to act as the electron sink. The research is designed in a way that makes it possible to track the movement of electrons and protons using combinations of variants with isotopic substitutions for substrates and solvent.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.