X-RAY CRYSTALLOGRAPHIC STUDIES OF METABOLIC ENZYMES

代谢酶的 X 射线晶体学研究

• 批准号: 8363559
• 负责人: STEVEN E EALICK
• 金额: $ 4.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363559
• 关键词: 2,6-Diaminopurine; Active Sites; Allantoin; Allopurinol; Anabolism; Antibiotics; Applications Grants; Biological Factors; Carboxy-Lyases; Complex; Data; Data Collection; Drug Delivery Systems; Drug Design; Energy Transfer; Enzymatic Biochemistry; Enzymes; Evolution; Funding; Future; Grant; Home environment; Hydrolase; Individual; Klebsiella pneumonia bacterium; Laboratories; Laboratory Study; Ligands; Longevity; Lyase; Malignant Neoplasms; Metabolic; Metabolic Pathway; Mus; N-methylacetamide-oxotremorine M; National Center for Research Resources; Nature; Nucleic Acids; Paper; Parasitic infection; Pathway interactions; Principal Investigator; Process; Protein Biosynthesis; Proteins; Publishing; Purine Nucleotides; Purines; Pyrimidine; Pyrimidine Nucleotides; Research; Research Infrastructure; Resources; Roentgen Rays; Signal Transduction; Source; Streptomyces; Structure; System; Testing; Texas; United States National Institutes of Health; Work; antitumor agent; azinomycin B; branched-chain-amino-acid transaminase; cost; design; enzyme structure; functional group; interest; member; metabolic abnormality assessment; programs; protein function; protein protein interaction; purine

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. Our laboratory studies the structures and functions of metabolic enzymes. We are interested in the catalytic mechanisms of individual enzymes, protein:protein interactions between enzymes within a pathway, evolution of protein function, and drug design. From the purine and pyrimidine metabolic pathways, monochromatic data will be taken on crystals of two enzymes from the newly discovered catabolic pathway from Klebsiella pneumoniae. KpHpxT 5-hydroxyisourate hydrolase (HIU) crystals that have been soaked with ligands allopurinol, 8-azaxanthine, allantoin, and 2,6-diaminopurine, and on KpHpxQ, 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase, crystals that have been soaked with the ligand allopurinol, will be tested to better characterize the active sites. Prior crystals of the former diffracted to 2 - 2.5 ¿ and the latter to 1.6  2 ¿. Five other enzymes in this catabolic pathway will be targets for later data collection trips. 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. In conjunction with our structural studies of the proteins involved in the biosynthesis of the purine-derived antibiotic toxoflavin, data will be taken on crystals of ToxA, which methylates demethlyated toxflavin, as well as TflA, an oxoflavin lyase, which degrades toxoflavin. Preliminary data from our home source indicates that the crystals diffract to better than 2 ¿. These studies are a first step in a long range program designed to understand the biosynthesis of purine-derived metabolites. In addition, toxoflavin biosynthesis is a good system to study N-N bond formation, a process found in a significant number of natural products for which the mechanistic enzymology is still poorly understood. For our studies of enzymes in the azinomycin B biosynthetic pathway, crystals of AziC1, a proposed branched-chain amino acid aminotransferase, from Streptomyces sahachiori will be tested for diffraction. No prior structures for enzymes in this pathway have been determined, so future data trips will involve additional pathway members as diffraction quality crystals are produced. Azinomycin B, a complex natural product isolated from Streptomyces griseofuscus, is a naturally occurring antibiotic that shows antitumor activity at nanomolar concentrations and increases life span in P388 leukemic mice. Because the functional groups found in azinomycin B are both unusual and densely assembled, it is of interest to develop an understanding of the biosynthetic strategy utilized in nature and exploit this to develop additional antitumor agents. The lab has published 18 papers on enzymes in the purine and pyrimidine metabolic pathways in conjunction with our collaborator Dr. Tadhg Begley, now at Texas A&M. This work is supported by NIH grant 5R01GM73220. The azinomyacin research represents a new direction for the laboratory in conjunction with Dr. Coran Watanabe of Texas A&M. We have recently submitted an R01 grant application to NIH in support of this project.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且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 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们的实验室研究代谢酶的结构和功能，我们对单个酶的催化机制、途径内酶之间的蛋白质与蛋白质相互作用、蛋白质功能的进化和药物设计感兴趣。 从嘌呤和嘧啶代谢途径中，将从肺炎克雷伯菌新发现的分解代谢途径中的两种酶的晶体中获取单色数据，这些酶已用配体别嘌呤醇、8-氮杂黄嘌呤、尿囊素浸泡。 ，和 2,6-二氨基嘌呤，以及 KpHpxQ，将测试已用配体别嘌呤醇浸泡的 2-氧代-4-羟基-4-羧基-5-脲基咪唑啉 (OHCU) 脱羧酶晶体，以更好地表征前者衍射至 2 - 2.5 的先前晶体。 ¿后者为 1.6 2 ¿该分解代谢途径中的其他五种酶将成为后续数据收集之旅的目标，嘧啶和嘌呤核苷酸是核酸合成的重要组成部分，并且由于其重要性也可以参与能量转移和存储、蛋白质合成和信号传导。在这些分子中，其代谢途径中的酶代表了治疗许多疾病（包括癌症和几种寄生虫感染）的潜在药物靶标。 结合我们对嘌呤衍生抗生素毒黄素生物合成所涉及的蛋白质的结构研究，我们将获取甲基化去甲基化毒黄素的 ToxA 晶体以及降解毒黄素的氧黄素裂解酶 TflA 的初步数据。来自我们国内的消息表明晶体的衍射强度优于 2 ¿这些研究是旨在了解嘌呤衍生代谢物生物合成的长期计划的第一步。此外，毒黄素生物合成是研究 N-N 键形成的良好系统，N-N 键形成是在大量天然产物中发现的过程。机械酶学仍然知之甚少。 为了我们对阿齐霉素 B 生物合成途径中的酶的研究，将对来自 Streptomyces sahachiori 的 AziC1（一种拟议的支链氨基酸氨基转移酶）晶体进行衍射测试，尚未确定该途径中酶的先前结构，因此未来的数据传输。随着衍射质量晶体的产生，阿齐霉素 B（一种从灰褐链霉菌中分离出来的复杂天然产物）将涉及其他途径成员。是一种天然存在的抗生素，在纳摩尔浓度下显示出抗肿瘤活性并延长 P388 白血病小鼠的寿命，因为阿齐霉素 B 中发现的官能团既不寻常又密集组装，因此了解阿齐霉素 B 中使用的生物合成策略很有意义。性质并利用它来开发更多的抗肿瘤药物。 该实验室与现在在德克萨斯农工大学的合作者 Tadhg Begley 博士一起发表了 18 篇关于嘌呤和嘧啶代谢途径中的酶的论文。这项工作得到了 NIH 拨款 5R01GM73220 的支持。阿齐霉素研究代表了该实验室的一个新方向。我们与德克萨斯农工大学的 Coran Watanabe 博士合作，最近向 NIH 提交了 R01 拨款申请以支持该项目。