STRUCTURE AND MECHANISM OF HOTDOG-FOD THIOESTERASES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 硫代酯是普遍存在的代谢物和调节剂，尤其与脂肪代谢有关，但不限于此。硫代酯是通过硫代酯酶的作用除去的，硫代酯酶催化水解反应生成母体羧酸和硫醇。这些酶中的大多数属于我们研究中的热狗硫代酯酶家族。热狗硫酯酶家族的功能是调节所有生命形式中的酰基辅酶A代谢物和酰基载体蛋白(ACPs)的水平。每种生物体内都有各种各样的热狗硫酯酶发挥作用(即人类有21种；铜绿假单胞菌有29种)。热狗的硫代酯酶首先被生物化学家定性为II型硫代酯酶，在脂肪酸和聚酮的合成中发挥作用。尽管热狗折叠硫代酯酶催化常见的化学反应，但它们显示了广泛的催化基序和底物识别元件。尽管这种多样性为特定的抑制剂提供了靶向优势，但它需要对各种家族成员进行鉴定、比较结构分析以及功能和催化机制的确定。 我们的计划是对选定的硫代酯酶进行详细的研究，以阐明控制底物识别和催化的基本原理，开发特定抑制剂的设计策略，并扩大我们对硫酯生物化学的知识。这项研究的目标是确定热狗硫代酯酶的生化功能、催化机制和紧密结合的抑制剂，这些酶与脂肪相关的人类遗传性疾病有关，是肥胖和脑癌的基础，也是细菌病原体对人类的毒力所必需的。我们的直接目标包括五种大肠杆菌热狗硫酯酶，YbdB，YbcG，YbaW，YicA，Ycel，它们也在一系列人类病原体中发现。(由NIH GM028688资助)。这个热狗结构域参与了许多细胞过程，从硫酯水解、脂肪酸生物合成的转录调控到芳香族化合物(如苯乙酸)的脱氢。“热狗折叠”由七个搁浅的反平行的贝塔折叠而成，被称为“小面包”，它包裹着一个五圈的阿尔法螺旋“香肠”。通过研究硫代酯酶超家族中的这个结构域，我们希望了解它作为一个普遍存在于所有生命分支中的基序所起的代谢作用。特别是，我们正在研究BH1999硫代酯酶、3-HBA-CoA硫代酯酶和PaaI硫代酯酶。

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. Thioesters are ubiquitous metabolites and regulators, especially relevant, but not limited, to lipid metabolism. Thioesters are removed through the action of thioesterases, which catalyze hydrolysis reactions to generate parent carboxylic acids and thiols. A majority of these enzymes belong to the hotdog thioesterase family targeted in our studies. The hotdog thioesterase family functions to regulate the levels of acyl-CoA metabolites and acyl-Acyl Carrier Proteins (ACPs) in all forms of life. A wide variety of hotdog thioesterases function within each organism (viz, 21 in humans; 29 in Pseudomonas aeruginosa). The hotdog thioesterases were first characterized by biochemists as type II thioesterases, functioning in fatty acid and polyketide synthesis. Despite the fact that they catalyze common chemical reactions, the hotdog fold thioesterases display a wide range of catalytic motifs and substrate recognition elements. Although this diversity provides a targeting advantage for specific inhibitors, it demands the identification, comparative structure analysis, and determination of function and catalytic mechanism for a variety of family members. Our plan is to carry out a detailed study of selected thioesterases in order to elucidate the underlying principles that govern substrate recognition and catalysis, develop a strategy for the design of specific inhibitors, and expand our knowledge of thioester biochemistry. The goal of the study is to determine biochemical functions, catalytic mechanisms and tight binding inhibitors for hotdog thioesterases that are implicated in lipid-related, human genetic disorders that underlie obesity and brain cancer, and that are required for virulence of bacterial pathogens for humans. Our immediate targets include the five E. coli hotdog thioesterases, YbdB, YbcG, YbaW, YicA, Ycil, which are also found in a range of human pathogens. (funded by NIH GM028688).This Hotdog domain is involved in many cellular processes which range from thiester hydrolysis, transcriptional regulation of fatty acid biosynthesis, to the degredation of aromatic compounds such as phenylacetic acid. The "Hotdog fold" consists of seven stranded antiparallel beta-sheets as the "bun", which wraps around a five-turn alpha-helical "sausage". By studying this domain within the thioesterase superfamily we hope to understand the metabolic role it plays as a ubiquitous motif found in all branches of life. In particular we are studying BH1999 thioesterase, 3-HBA CoA thioesterase, and PaaI thioesterase.