Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, 3052 Australia; School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.
School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.
J Struct Biol. 2020 Apr 1;210(1):107477. doi: 10.1016/j.jsb.2020.107477. Epub 2020 Feb 4.
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.
酰基辅酶 A 硫酯酶 (ACTS) 催化脂肪酸链和辅酶 A (CoA) 之间硫酯键的水解，生成游离脂肪酸链和 CoA。这些酶在原核生物和真核生物中广泛表达，在脂质代谢中发挥重要作用。有 25 种硫酯酶家族，根据其活性位点结构、蛋白质寡聚化和底物特异性进行细分。了解这些家族内的调节机制很重要，因为它们在控制各种脂肪酸和 CoA 结合化合物的细胞浓度方面发挥作用。在这里，我们报告了一种来自金黄色葡萄球菌的 ACT 的新型抑制模式的结构基础。该酶显示出热狗折叠结构，由五个β-链包裹在中央α-螺旋周围，其 C 末端还有一个额外的 30 个残基α-螺旋。我们表明该酶是一个六聚体，对丁酰 CoA 具有特异性。结构分析揭示了可能的催化残基，我们通过定点突变表明，Asn28、Asp43 和 Thr60 对活性至关重要。此外，我们表明 Asn28Ala 使酶的寡聚状态不稳定，形成两个不同的群体。该酶与底物丁酰 CoA 共结晶产生了一个晶体，其中三个 CoA 配体结合在酶的活性位点：CoA、丁酰 CoA 和二硫代 CoA，后者抑制酶活性。我们的研究为六聚体硫酯酶的结构和特异性、抑制反馈机制以及在短链脂肪酸生产（如生物燃料、药物和工业化合物）中的可能生物技术应用提供了新的见解。
