Flavoenzymes in Pyrimidine Metabolism

嘧啶代谢是至关重要的，这使得在化学水平上了解它变得重要，并使其成为药物开发的极佳靶点。我们将研究二氢罗酸脱氢酶(DHOD)、生物合成途径中的黄素依赖酶以及进化上相关的二氢尿苷合成酶(DUSS)的反应机制，这些酶在tRNA成熟过程中减少特定的尿嘧啶。我们的目标是从描述过渡态到揭示催化中的动态行为，来阐明底物或配体专一性的反应机制和来源。这些研究的结果将有助于酶抑制剂的设计，这些酶抑制剂可能被开发成有用的候选药物。过渡态结构是酶催化的核心。以前，我们发现在第1A类和第2类DHOD中黄素还原的过渡态之间存在显著差异，这表明需要更高水平的理解。通过测量~(13)C和~(15)N的动力学同位素效应，研究了三类DHOD中黄素还原的过渡态。停流实验将被用来确定氚同位素对减少1B级DHOD的影响。对1B类DHOD的补充性停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专门与1A类DHOD结合的抑制剂，这种DHOD存在于一些病原菌和原生动物中。我们的动力学和结构研究表明需要合成和研究一种新的分子。然而，我们的抑制剂不与2类酶结合的原因仍然是一个谜。随机突变将被用来产生不再被抑制的功能1A类突变体，反之，产生被抑制的2类突变体。有趣的酶将在热力学、动力学和结构上进行详细研究。相关的化学是由DUSS执行的，黄素蛋白在结构上与DHOD相关，并在成熟的tRNA中减少特定的尿嘧啶部分。二氢尿嘧啶的功能仍不确定，但它的广泛出现表明了它的重要作用，最近它在肺癌中被证明是重要的。我们将确定选定的Duss模型的底物特异性，并通过化学和生物物理手段探索蛋白质与tRNA的相互作用。 当某些蛋白质加速化学反应时，维生素B2就会传递电子，这些化学反应会产生或修改DNA或RNA的组成成分，而DNA或RNA是携带遗传信息的分子。专门干扰感染细菌中这些重要反应的化合物可以被用作药物。为了设计这样的化合物，我们将通过观察与维生素相关的颜色变化来非常详细地研究几种蛋白质的反应。我们对化学反应速度的研究将确定蛋白质的重要部分，它们在反应中如何移动，它们如何加速产品的合成，以及这些反应可能如何被阻止。

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.