PURINE AND PYRIMIDINE METABOLISM

该子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心赠款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 嘧啶和嘌呤核苷酸是合成核酸的基本构件，也可以参与能量转移和储存、蛋白质合成和信号传导。由于这些分子的重要性，其代谢途径中的酶代表了用于治疗许多疾病的潜在药物靶标，包括癌症和几种类型的寄生虫感染。 我们已经进行了各种酶的结构研究，这些酶在嘧啶和嘌呤的代谢中发挥作用。腺苷激酶（Adenosine kinase，AK）是寄生虫嘌呤代谢的关键酶，是弓形虫感染的潜在化疗靶点，可催化ATP依赖的腺苷磷酸化。嘌呤核苷磷酸化酶（PNP）是一种重要的嘌呤核苷酸抢救酶，催化核糖核苷和2 '-脱氧核糖核苷可逆地磷酸化为游离碱和（2'-脱氧）核糖-1-磷酸。 RutA是一种FMN依赖性单加氧酶，参与最近发现的嘧啶降解途径，在E.杆菌尿苷磷酸化酶（UP）催化尿苷的可逆磷酸解，形成核糖-1-磷酸和尿嘧啶。乳清酸核苷-5 '-磷酸脱羧酶乳清酸磷酸核糖基转移酶（OMPDC-OPRT）是催化尿苷-5'-单磷酸（UMP）合成的最后两步的双功能酶。 除了它们的许多细胞用途之外，一些生物体可以代谢核苷酸作为氮源。最近两个研究小组对克雷伯氏菌的研究揭示了一个基因簇，该基因簇负责表达利用嘌呤作为该生物体唯一氮源的酶。为了更好地理解这一有趣的途径，我们对肺炎克雷伯氏菌中催化次黄嘌呤分解为尿囊素的几种酶进行了结构表征。 假尿苷是尿苷的C-糖苷异构体，是RNA中最丰富的修饰。它广泛存在于tRNA、rRNA、snRNA和snoRNA中。 最近，各种生物化学，生物物理和遗传学研究的特点是两种酶，主要负责假尿苷的生物合成。 假尿苷激酶（YeiC）将假尿苷磷酸化为假尿苷-5 '-磷酸，假尿苷糖苷酶（YeiN）通过切割C-C糖苷键催化从假尿苷-5'-磷酸转化为尿苷和核糖-5-磷酸。 这些酶的结构研究将提供深入了解假尿苷生物合成的机制，并提供工具，筛选可能的抗菌药物，针对这一途径。 米多霉素是一种肽基核苷抗生素，对植物上的白粉病具有很强的活性，并在商业上用作杀真菌剂。 米多霉素生物合成的初始步骤涉及酶MilB在糖苷键处切割5-羟甲基胞苷-5 '-单磷酸。 已经表明，MilB还催化胞苷-5 '-单磷酸的裂解反应。 解决了MilB的晶体结构，以更好地了解其结构如何与其他核苷酸水解酶不同，以及它如何提供其底物特异性。 毒黄素是一种氮杂蝶啶，对许多植物、真菌、动物和细菌有毒。最近，毒黄素引起了越来越多的关注，因为水稻植物被产毒黄素的细菌（例如颖壳伯克霍尔德氏菌）感染，导致美国和亚洲的水稻作物遭受重大损失。 毒黄素生物合成中的几个步骤涉及未表征的蛋白质，这些蛋白质可能具有新的化学性质。具体地，蛋白ToxC和/或ToxD似乎催化氮-氮键形成，这是知之甚少的，并且预测蛋白ToxA在毒黄素生产的最后步骤中催化顺序的甲基转移反应。 为了对抗毒黄素的毒性，最近的工作已经开发了一些酶在许多脂肪族和芳香族化合物的生物合成和生物降解中利用分子氧的能力。 最近的工作已经导致成功生产转基因水稻植物，其表达多粘类芽孢杆菌JH 2的推定的加氧酶毒黄素裂解酶（TflA），以对抗产毒黄素细菌对水稻的有害影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Pyrimidine and purine nucleotides are essential building blocks for the synthesis of nucleic acids and can also take part in energy transfer and storage, protein synthesis and signaling. Because of the importance of these molecules, the enzymes in their metabolic pathways represent potential drug targets for the treatment of many conditions including cancer and several types of parasitic infections. We have undertaken structural studies of various enzymes that play roles in the metabolism of pyrimidines and purines. Adenosine kinase (AK), a key enzyme in purine metabolism in parasites and a potential chemotherapeutic target for the treatment of Toxoplasma gondii infections, catalyzes the ATP dependent phosphorylation of adenosine. Purine nucleoside phosphorylase (PNP), which catalyzes the reversible phosphorolysis of ribonucleosides and 2'- deoxyribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate, is an important enzyme for the salvage of purine nucleotides. RutA is a FMN dependent mono-oxygenase involved in a recently discovered pyrimidine degradation pathway that converts uracil (or thymine) to 3-hydroxypropionate (or 2-methyl-3-hydroxypropionate) in E. coli. Uridine phosphorylase (UP) catalyzes the reversible phosphorolysis of uridine with the formation of ribose-1-phosphate and uracil. Orotidine-5'-phosphate decarboxylase orotate phosphoribosyltransferase (OMPDC-OPRT) is a bifunctional enzyme that catalyzes the last two steps in the synthesis of uridine-5'-monophosphate (UMP). In addition to their many cellular uses, some organisms can metabolize nucleotides as a nitrogen source. Recent studies by two groups on Klebsiella sp. have revealed a gene cluster that is responsible for expressing the enzymes for utilizing purines as a sole nitrogen source in this organism. We have structurally characterized several of the enzymes that catalyze the breakdown of hypoxanthine to allantoin in Klebsiella pneumoniae in order to better understand this interesting pathway. Pseudouridine is the C-glycoside isomer of uridine and is the most abundant modification in RNA. It ubiquitously exists in tRNA, rRNA, snRNA and snoRNA. Recently, various biochemical, biophysical and genetic studies characterized two enzymes that are predominantly responsible for the biosynthesis of pseudouridine. Pseudouridine kinase (YeiC) phosphorylates pseudouridine to pseudouridine-5'-phosphate and pseudouridine glycosidase (YeiN) catalyzes the conversion from pseudouridine-5'-phosphate to uridine and ribose-5-phosphate by cleaving the C-C glycosidic bond. Structural studies of these enzymes will provide insights into the mechanism of pseudouridine biosynthesis and provide tools for the screening of possible antibacterial drugs that target this pathway. Mildiomycin is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease on plants and is used commercially as a fungicide. The initial steps of mildiomycin biosynthesis involve the cleavage of 5-hydroxymethyl cytidine-5'-monophosphate at the glycosidic bond by the enzyme MilB. It has been shown that MilB also catalyzes the cleavage reaction for cytidine-5'-monophosphate. The crystal structure of MilB was solved to better understand how its structure varies from other nucleotide hydrolases and how it provides its substrate specificity. Toxoflavin is an azapteridine that is poisonous to many plants, fungi, animals, and bacteria. Recently, toxoflavin has gained increasing interest because infection of rice plants by toxoflavin-producing bacteria such as Burkholderia glumae has led to a substantial loss of rice crops in the United States and Asia. Several of the steps in the biosynthesis of toxoflavin involve uncharacterized proteins that may potentially exhibit novel chemistry. Specifically, the proteins ToxC and/or ToxD appear to catalyze nitrogen-nitrogen bond formation which is poorly understood and the protein ToxA is predicted to catalyze sequential transmethylation reactions in the final step of toxoflavin production. To combat the toxicity of toxoflavin, recent work has exploited the ability of some enzymes to utilize dioxygen in the biosynthesis and biodegradation of numerous aliphatic and aromatic compounds. Recent work has led to the successful production of transgenic rice plants which express the putative oxygenase toxoflavin lyase (TflA) of Paenibacillus polymyxa JH2 to combat the deleterious effects of toxoflavin-producing bacteria on rice.