Flavoenzymes in Pyrimidine Metabolism

描述（由申请人提供）：嘧啶代谢是至关重要的，因此在化学水平上理解它很重要，并使其成为药物开发的绝佳靶点。我们将研究生物合成途径中黄素依赖性酶——二氢乙酸脱氢酶（DHODs）和进化相关的二氢吡啶合成酶（DUSs）的反应机制，这些酶在tRNA成熟过程中减少特定的尿嘧啶。我们的目标是阐明反应机制和底物或配体特异性的来源，从表征过渡态到揭示催化中的动态行为。这些研究结果将有助于酶抑制剂的设计，并可能开发成有用的候选药物。过渡态结构是酶催化的核心。之前我们发现，1A类DHODs和2类DHODs中黄素还原的过渡状态存在显著差异，这表明需要更高水平的理解。通过测量13C和15N的动力学同位素效应，探讨了三类DHODs中黄素还原的过渡态。停流实验将用于确定氘同位素对1B类DHOD还原的影响。对1B类DHOD的互补止流和单分子研究将使我们能够剖析控制黄素化学，分子内电子转移和动力学的因素。我们已经发现了两种特异性结合1A类DHODs的抑制剂，这种抑制剂存在于一些致病菌和原生动物中。我们的动力学和结构研究表明一种新的分子有待合成和研究。然而，我们的抑制剂不与2类酶结合的原因仍然是一个谜。随机诱变将用于创建不再被抑制的功能性1A类突变体，反之，2类突变体被抑制。有趣的酶将详细研究热力学，动力学和结构。相关的化学反应由dus完成，dus是一种与DHODs结构相关的黄蛋白，可以在成熟的tRNA中减少特定的尿嘧啶部分。二氢尿嘧啶的功能尚不清楚，但它的广泛存在表明它在肺癌中起着重要作用，最近已证明它在肺癌中很重要。我们将确定选定模型DUSs的底物特异性，并通过化学和生物物理手段探测蛋白质与tRNA的相互作用。

DESCRIPTION (provided by applicant): Pyrimidine metabolism is vital, making it important to understand at the chemical level and making it an excellent target for drug development. We will investigate the reaction mechanisms of dihydroorotate dehydrogenases (DHODs), flavin-dependent enzymes in the biosynthetic pathway, and the evolutionarily related dihydrouridine synthases (DUSs), which reduce specific uracils during the maturation of tRNA. Our goal is to elucidate reaction mechanisms and origins of substrate or ligand specificity in ways ranging from characterizing transition states to uncovering dynamic behavior in catalysis. The results of these studies will facilitate the design of enzyme inhibitors, which may be developed into useful drug candidates. Transition state structures are at the heart of enzymatic catalysis. Previously we found significant differences between the transition states for flavin reduction in Class 1A and Class 2 DHODs, indicating the need for a higher level of understanding. The transition states for flavin reduction in the three classes of DHODs will be probed by measuring 13C and 15N kinetic isotope effects. Stopped-flow experiments will be used to determine deuterium isotope effects on the reduction of a Class 1B DHOD. Complementary stopped-flow and single-molecule studies on a Class 1B DHOD will enable us to dissect factors controlling the chemistry at the flavins, intramolecular electron transfer, and dynamics. We have already discovered two inhibitors that bind specifically to Class 1A DHODs which occurs in some pathogenic bacteria and protozoa. Our kinetic and structural studies suggest a new molecule to be synthesized and studied. However, the reason that our inhibitors do not bind to Class 2 enzymes remains an enigma. Random mutagenesis will be used to create functional Class 1A mutants that are no longer inhibited, and conversely, Class 2 mutants that are inhibited. Interesting enzymes will be studied in detail thermodynamically, kinetically, and structurally. Related chemistry is performed by the DUSs, flavoproteins which are structurally related to DHODs and reduce specific uracil moieties in maturing tRNA. The function of dihydrouracil remains uncertain, but its widespread occurrence suggests an important role, and it has recently been shown to be important in lung cancer. We will determine the substrate specificities of selected model DUSs and probe the interactions of the protein and tRNA by chemical and biophysical means. Vitamin B2 transfers electrons when certain proteins speed chemical reactions that create or modify the building-blocks of DNA or RNA the molecules which carry genetic information. Compounds that specifically interfere with these vital reactions in infectious bacteria could be used as drugs. In order to design such compounds, we will study the reactions of several proteins at a very high level of detail by observing the color changes associated with the vitamin. Our studies of the rates of the chemical reactions will identify important parts of the proteins, how they move during reactions, how they speed the synthesis of products, and how these reactions might be blocked.