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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 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理感兴趣，蛋白质：途径内酶之间的蛋白质相互作用，蛋白质功能的演变和药物设计。 从嘌呤和嘧啶代谢途径中，将对来自克雷伯氏菌肺炎的新发现分解代谢途径的两种酶的晶体进行单色数据。 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-ureidomidazoline (OHCU) decarboxylase, crystals that have been soaked with配体素素醇将进行测试，以更好地表征活性位点。前者的先前晶体被衍射为2-2.5？，后者到1.6 2»该分解代谢途径中的其他五种酶将成为以后的数据收集旅行的目标。嘧啶和嘌呤核苷酸是合成核酸的必不可少的基础，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治疗许多疾病（包括癌症和几种类型的寄生虫感染）的潜在药物靶标。 结合我们对纯衍生的抗生素毒素类黄酮的生物合成涉及的蛋白质的结构研究，将对毒Toxa的晶体进行数据，这些毒素甲基化的甲基化甲基化的毒tflavin以及TFLA，以及一种脱脂液酶，脱脂酶，脱脂剂，脱脂剂，脱脂。来自我们家用来源的初步数据表明，晶体衍射效果要大于2。这些研究是远距离计划的第一步，旨在了解纯代谢物的生物合成。此外，毒素生物合成是研究N-N键形成的好系统，这是在许多天然产物中发现的过程，其机械酶学仍然对此知之甚少。 为了我们研究偶氮霉素B生物合成途径中的酶，将测试来自Sahachiori链霉菌的Azic1的晶体，Azic1的晶体，一种提议的分支链氨基酸氨基酸氨基转移酶。尚未确定该途径中酶的先前结构，因此随着衍射质量晶体的产生，将来的数据旅行将涉及其他途径成员。偶氮霉素B是一种从链霉菌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抗肿瘤活性，并在p388白血病小鼠中增加寿命。由于在苯霉素B中发现的官能团既不寻常且纯净地组装，因此有意义地了解自然界中使用的生物合成策略并利用它来开发其他抗肿瘤剂。 该实验室与我们的合作者Tadhg Begley博士（现在位于德克萨斯州的A＆M）发表了有关采购和嘧啶代谢途径的18篇论文。这项工作得到了NIH授予5R01GM73220的支持。偶氮霉素研究代表了实验室与德克萨斯州A＆M的Coran Watanabe博士结合的新方向。我们最近向NIH提交了R01赠款申请，以支持该项目。