Acyl-coenzyme A thioesterases (ACTs) catalyse the hydrolysis of thioester bonds between fatty-acyl chains and coenzyme A (CoA), producing a free fatty-acyl chain and CoA. These enzymes are expressed ubiquitously across prokaryotes and eukaryotes, and play important roles in lipid metabolism. There are 25 thioesterase families, subdivided based on their active site configuration, protein oligomerization, and substrate specificity. Understanding the mechanism of regulation within these families is important due to their roles in controlling the cell concentration of a range of fatty acids and CoA-bound compounds. Here we report a structural basis for a novel mode of inhibition of an ACT from Staphylococcus aureus. The enzyme displays a hotdog fold composed of five β-strands wrapping around a central α-helix, and an additional 30 residue α-helix located at its C-terminus. We show that the enzyme is a hexamer and has specificity towards butyryl-CoA. Structural analysis revealed putative catalytic residues, and we show through site directed mutagenesis that Asn28, Asp43, and Thr60 are critical for activity. Additionally, we show that the Asn28Ala destabilises the enzyme oligomeric state into two distinct populations. Co-crystallization of the enzyme with the substrate butyryl-CoA produced a crystal with three CoA ligands bound in the enzyme active sites: CoA, butyryl-CoA, and disulphide-CoA, the latter of which inhibits enzyme activity. Our study provides new insights into the structure and specificity of hexameric thioesterases, inhibitory feedback mechanisms, and possible biotechnological applications in short-chain fatty acid production such as biofuels, pharmaceuticals, and industrial compounds.

中文翻译：

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二硫化物-CoA抑制丁酰-CoA六聚硫酯酶的结构基础。

酰基辅酶A硫酯酶（ACTs）催化脂肪酰基链和辅酶A（CoA）之间的硫酯键水解，产生游离的脂肪酰基链和CoA。这些酶在原核生物和真核生物中普遍表达，并在脂质代谢中起重要作用。有25个硫酯酶家族，根据其活性位点配置，蛋白质寡聚和底物特异性进行细分。了解这些家族中的调节机制非常重要，因为它们在控制一系列脂肪酸和与CoA结合的化合物的细胞浓度中的作用。在这里，我们报告抑制金黄色葡萄球菌的ACT的新型模式的结构基础。该酶显示出由围绕中央α螺旋缠绕的5条β链组成的热狗折叠，在其C末端还有30个残基α-螺旋。我们表明该酶是六聚体，对丁酰辅酶A具有特异性。结构分析显示推定的催化残基，并且我们通过定点诱变显示Asn28，Asp43和Thr60对活性至关重要。此外，我们表明，Asn28Ala破坏了酶寡聚状态，变成两个不同的群体。酶与底物丁酰辅酶A的共结晶产生了晶体，该晶体具有在酶活性位点结合的三个CoA配体：CoA，丁酰辅酶A和二硫化物CoA，后者抑制酶的活性。我们的研究为六聚硫酯酶的结构和特异性，抑制性反馈机制，