Structural Biology of Purine and Pyrimidine Biosynthesis and Metabolism

描述(申请人提供)：本申请的总体目标是研究参与嘌呤和嘧啶生物合成的酶的结构和功能，嘌呤和嘧啶的分解代谢，以及嘌呤在细胞代谢物生物合成中的利用。参与嘌呤和嘧啶生物合成和代谢的酶是包括癌症、细菌感染和寄生虫感染在内的多种人类疾病的药物设计目标。虽然大多数的嘌呤和嘧啶生物合成酶的结构已经被弄清楚，但只有少数结构是高等生物中出现的多功能酶的已知结构。这些多功能酶反过来可能有助于理解嘌呤小体，这是一种可能无法进行结晶学分析的大型多蛋白质生物合成复合体。拟议的研究包括脊椎动物三功能酶PurD-PurM-Purn，它催化嘌呤生物合成的步骤2、3和5，以及在其他高等生物中发现的变体PurD-PurM和PurD-PurM-PurM4-Purn。我们还建议从疟疾寄生虫中研究OMPDC-OPRT，它催化嘧啶生物合成的步骤6和5。以前已经描述了降解嘌呤和嘧啶的分解代谢途径，关键酶的结构也是可用的；然而，最近在肺炎克雷伯菌中发现了一条生物合成嘧啶的新途径，并在大肠杆菌中发现了一条降解嘧啶的新途径。这些新途径的发现令人惊讶，而且大多数基因产物在生化和结构上都没有特征。生物信息学表明，黄素酶催化嘧啶分解代谢途径中的开环，这是黄素酶学中的一个新的催化基序。嘌呤分解代谢操纵子编码两种新的酶活性--推测的黄素依赖的尿酸酶和铁依赖的黄嘌呤氧化酶。最后，对现有基因组的分析表明，许多操纵子与未表征的环水解酶催化的开环反应有关。这些反应通常与嘌呤衍生的代谢物有关，如叶酸、核黄素和钼蝶呤，这表明嘌呤在生物合成额外代谢物方面得到了广泛的利用。我们将通过检测毒黄素的生物合成来开始研究嘌呤的利用，毒黄素是理解N-N键形成的机制的一个很好的系统。这项研究所需的大多数酶都已被克隆和过度表达。我们将使用X射线结晶学来确定这些酶的结构，并与Tadhg Begley教授合作研究机械酶学。与公共卫生相关：嘌呤和嘧啶核苷酸是DNA和RNA的基石。所有形式的生命都依赖于这些分子，它们在细胞中的水平受到生物合成、输入、降解和新陈代谢的调节。了解这些过程为抗癌和抗微生物化疗提供了可能的靶点。

DESCRIPTION (provided by applicant): The overall objective of this application is to study the structures and functions of enzymes involved in purine and pyrimidine biosynthesis, purine and pyrimidine catabolism, and purine utilization in the biosynthesis of cellular metabolites. Enzymes involved in purine and pyrimidine biosynthesis and metabolism serve as targets for drug design in wide arrange of human diseases including cancer, bacterial infections and parasitic infections. While most of the structures for individual purine and pyrimidine biosynthetic enzymes have been worked out, only a few structures are known for multifunctional enzymes that occur in higher organisms. The multifunctional enzymes may in turn aid in understanding the purinosome, a large multiprotein biosynthetic complex that probably is not amenable to crystallographic analysis. Proposed studies include the vertebrate trifunctional enzyme PurD-PurM-PurN, which catalyzes steps 2, 3 and 5 of purine biosynthesis, and PurD-PurM and PurD-PurM-PurM4-PurN, which are variants found in other higher organisms. We also propose to study OMPDC-OPRT from malaria parasite, which catalyzes steps 6 and 5 of pyrimidine biosynthesis. Catabolic pathways for the degradation of purines and pyrimidines have been previously described and structures are available for the key enzymes; however, recently a new pathway for pyrimidine biosynthesis was discovered in Klebisella pneumoniae and a new pathway was discovered for pyrimidine degradation in Escherichia coli. The discovery of these new pathways was surprising and most of the gene products are both biochemically and structurally uncharacterized. Bioinformatics suggest that a flavoenzyme catalyzes the ring opening in the pyrimidine catabolic pathway - a new catalytic motif in flavoenzymology. The purine catabolic operon encodes two novel enzymatic activities - a putative flavin- dependent uricase and an iron-dependent xanthine oxidase. Finally, analysis of the available genomes indicates many operons associated with uncharacterized cyclohydrolase-catalyzed ring-opening reactions. These reactions are usually associated with purine-derived metabolites such as folate, riboflavin and molypdopterin, suggesting a widespread utilization of purines in the biosynthesis of additional metabolites. We will begin to study purine utilization by examining the biosynthesis of toxoflavin, a good system for understanding the mechanistic enzymology of N-N bond formation. Most of the enzymes required for this research have been cloned and overexpressed. We will determine the structures of these enzymes using X-ray crystallography and in collaboration with Prof. Tadhg Begley study the mechanistic enzymology. PUBLIC HEALTH RELEVANCE: Purine and pyrimidine nucleotides are the building blocks for DNA and RNA. All forms of life depend on these molecules and their levels in the cell are regulated by biosynthesis, import, degradation and metabolism. Understanding these processes offers possible targets for both anticancer and antimicrobial chemotherapies.