CRYSTALLOGRAPHIC STUDIES OF CONFORMATIONAL CHANGES IN ADENYLATE-FORMING ENZYMESE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们实验室的工作是研究两组酶，A）一个大家族的腺苷酸形成酶，包括酰基辅酶A合成酶/连接酶和非核糖体肽合成酶（NRPS）的腺苷酸化结构域，以及B）参与细菌铁载体合成的其他NRPS相关酶。 腺苷酸形成酶催化两步反应：初始腺苷酸化反应以活化羧酸底物，随后是第二硫酯形成反应，其中形成酰基-CoA或酰基-泛酰巯基乙胺硫酯。 我们已经确定了这个酶家族的成员在两个构象，表现出戏剧性的140度的结构域旋转之间的两个半反应的结构。 我们正在进一步研究这一点，以探索仅催化腺苷酸化半反应的酶（来自M. thermoautotrophicus），以及研究该家族的成员，可能作为抗生素的目标（MbtA）。 在与Courtney Aldrich博士的合作中，我们将确定MbtA与他设计和合成的许多小分子抑制剂结合的结构。 我们有2.0A的AAE的本地数据，并将在CHESS收集SeMet标记蛋白质晶体的数据。 我们已经使MbtA结晶，但是这些处于表征的早期阶段。 我们工作的第二个领域涉及参与由NRPS和相关酶合成的其他铁载体（铁螯合）化合物的其他酶。 我们已经确定了参与绿脓菌荧光素合成的两种蛋白质的结构，绿脓菌荧光素是来自囊性纤维化相关病原体铜绿假单胞菌的铁载体。 其中一个是使用国际象棋的数据解决的。 这些结构是鉴定绿脓菌荧光素的合成途径和鉴定可阻断绿脓菌荧光素产生的抗感染剂的潜在靶标的更大结构/功能的一部分。 类似地，我们具有关于这些绿脓菌荧光素合成酶之一PvcA的边际天然数据（2.6A），并且计划在所请求的行程中收集改进的天然以及Se MAD数据。 我们已经确定了天然结构的其他几个样品将进行研究，以获得额外的配体结构，如样本部分（#5）所述。

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).