CRYSTALLOGRAPHIC STUDIES OF CONFORMATIONAL CHANGES IN ADENYLATE-FORMING ENZYMESE

腺苷酸形成酶构象变化的晶体学研究

• 批准号: 7721304
• 负责人: ANDREW M GULICK
• 金额: $ 2.72万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721304
• 关键词: Acyl Coenzyme A; Anti-Infective Agents; Antibiotics; Binding; Coenzyme A Ligases; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Cystic Fibrosis; Data; Enzymes; Family; Family member; Funding; Grant; Institution; Iron; L-Selenomethionine; Label; Ligands; Ligase; Molecular Conformation; Numbers; Pantetheine; Pathway interactions; Production; Proteins; Pseudomonas aeruginosa; Reaction; Research; Research Personnel; Resources; Rotation; Sampling; Siderophores; Source; Specimen; Staging; Structure; United States National Institutes of Health; Work; adenylate; carboxylate; design; improved; inhibitor/antagonist; member; pathogen; peptide synthase; pyoverdin; small molecule; synthetic enzyme; thioester

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Work in our lab is studying two groups of enzymes, A) adenylate-forming enzymes of a large family that includes acyl-CoA synthetases/ligases and adenylation domains of non-ribosomal peptide synthetases (NRPSs) as well as B) additional NRPS-related enzymes involved in bacterial siderophore synthesis. The adenylate-forming enzymes catalyze a two-step reaction: an initial adenylation reaction to active a carboxylate substrate followed by a second thioester-forming reaction in which the acyl-CoA or acyl-pantetheine thioester is formed. We have determined structures of members of this enzyme family in two conformations that demonstrate a dramatic 140-degree domain rotation between the two half-reactions. We are examining this further to explore enzymes that catalyze only the adenylation half-reaction (AAE from M. thermoautotrophicus) as well as studying members of this family that may serve as antibiotic targets (MbtA). In a collaboration with Dr. Courtney Aldrich, we will determine the structure of MbtA bound to a number of small molecule inhibitors that he has designed and synthesized. We have native data to 2.0A for AAE and will collect data on SeMet labeled protein crystals at CHESS. We have crystallized MbtA however these are in early stages of characterization. The second field of our work concerns other enzymes involved in other siderophore (iron-sequestering) compounds synthesized by NRPSs and related enzymes. We have determined structures of two proteins involved in the synthesis of pyoverdine, a siderophore from the Cystic Fibrosis-related pathogen, P. aeruginosa. One was solved using data from CHESS. These structures are part of a larger structure/function to identify the synthetic pathway for pyoverdine and to identify potential targets for anti-infective agents that could block pyoverdine production. Similarly, we have marginal native data (2.6A) on one of these pyoverdine synthetic enzyme, PvcA, and plan to collect improved native, as well as Se MAD data on the requested trip. Several other samples, for which we have determined native structures, will be studied to obtain additional liganded structures, as described in the Specimen Section (#5).

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们实验室的工作是研究两组酶，A)腺苷形成酶，包括酰辅酶A合成酶/连接酶和非核糖体多肽合成酶(NRPS)的腺化结构域，以及B)参与细菌铁载体合成的额外NRPS相关酶。腺苷形成酶催化两步反应：第一步腺化反应以激活羧酸盐底物，然后第二步硫酯形成反应，在该反应中形成酰基-辅酶A或酰基泛氨酸硫酯。我们已经确定了该酶家族成员在两种构象中的结构，这两种构象显示了两个半反应之间戏剧性的140度结构域旋转。我们正在进一步研究这一点，以探索只催化腺化半反应的酶(来自热自养分枝杆菌的AAE)，以及研究这个家族中可能作为抗生素靶标的成员(MBTA)。在与考特尼·奥尔德里奇博士的合作下，我们将确定MBTA与他设计和合成的一些小分子抑制剂结合的结构。我们有AAE的原始数据到2.0A，并将在国际象棋中收集关于SEMET标记的蛋白质晶体的数据。我们已经结晶了MBTA，然而，这些还处于表征的早期阶段。 我们工作的第二个领域涉及由NRPS和相关酶合成的其他铁载体化合物(铁隔离)所涉及的其他酶。我们已经确定了参与合成吡喃佛丁的两个蛋白质的结构，吡喃佛丁是囊性纤维化相关病原体铜绿假单胞菌的铁载体。其中一个是用国际象棋中的数据解决的。这些结构是一个更大的结构/功能的一部分，以确定吡喃佛定的合成途径，并确定可能阻止吡喃佛定生产的抗感染剂的潜在靶点。同样，我们有这些吡喃佛丁合成酶之一PvcA的边际原生数据(2.6A)，并计划在请求的行程中收集改进的原生数据以及SMAD数据。我们已经确定了天然结构的其他几个样品将被研究，以获得额外的连接结构，如样品部分(#5)所述。