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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 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。 本实验室主要研究代谢酶的结构和功能。 我们感兴趣的是单个酶的催化机制，蛋白质：蛋白质之间的相互作用，蛋白质功能的进化和药物设计。 从嘌呤和嘧啶代谢途径，单色数据将采取晶体的两种酶从新发现的分解代谢途径，从肺炎克雷伯氏菌。将测试已用配体别嘌呤醇、8-氮杂黄嘌呤、尿囊素和2，6-二氨基嘌呤浸泡的KpHpxT 5-羟基异尿酸水解酶（HIU）晶体，以及已用配体别嘌呤醇浸泡的KpHpxQ 2-氧代-4-羟基-4-羧基-5-脲基咪唑啉（OHCU）脱羧酶晶体，以更好地表征活性位点。前者的先前晶体衍射到2 - 2.5 <$，后者衍射到1.6  2分。这一分解代谢途径中的其他五种酶将成为以后数据收集旅行的目标。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。 结合我们的结构研究的蛋白质参与的嘌呤衍生的抗生素toxoflavin的生物合成，数据将采取的晶体ToxA，甲基化脱甲基toxflavin，以及TflA，oxoflavin裂解酶，降解toxoflavin。从我们的家庭来源的初步数据表明，晶体的作用优于2美分。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，毒黄素的生物合成是一个很好的系统来研究N-N键的形成，一个过程中发现的大量的天然产物的机制酶学仍然知之甚少。 对于我们在azinomycin B生物合成途径中的酶的研究，将测试来自Streptomyces sahachiori的AziC 1晶体的衍射，AziC 1是一种拟议的支链氨基酸氨基转移酶。在这一途径中没有酶的先前结构已经确定，因此未来的数据行程将涉及额外的途径成员，因为产生了衍射质量的晶体。 Azinomycin B是一种从灰褐链霉菌中分离的复杂天然产物，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。因为在azinomycin B中发现的官能团是不寻常的并且是密集组装的，所以对自然界中使用的生物合成策略进行理解并利用其开发另外的抗肿瘤剂是有意义的。 该实验室已经发表了18篇关于嘌呤和嘧啶代谢途径中的酶的论文，与我们的合作者Tadhg Begley博士合作，他现在在德克萨斯A&M。这项工作得到了NIH资助5 R 01 GM 73220的支持。azinomyacin的研究代表了与德克萨斯A&M的Coran Watanabe博士合作的实验室的新方向。我们最近向NIH提交了R 01资助申请，以支持该项目。