Department of Biochemistry & Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Nat Commun. 2023 Apr 19;14(1):2247. doi: 10.1038/s41467-023-37986-9.
ATP citrate lyase (ACLY) is the predominant nucleocytosolic source of acetyl-CoA and is aberrantly regulated in many diseases making it an attractive therapeutic target. Structural studies of ACLY reveal a central homotetrameric core citrate synthase homology (CSH) module flanked by acyl-CoA synthetase homology (ASH) domains, with ATP and citrate binding the ASH domain and CoA binding the ASH-CSH interface to produce acetyl-CoA and oxaloacetate products. The specific catalytic role of the CSH module and an essential D1026A residue contained within it has been a matter of debate. Here, we report biochemical and structural analysis of an ACLY-D1026A mutant demonstrating that this mutant traps a (3S)-citryl-CoA intermediate in the ASH domain in a configuration that is incompatible with the formation of acetyl-CoA, is able to convert acetyl-CoA and OAA to (3S)-citryl-CoA in the ASH domain, and can load CoA and unload acetyl-CoA in the CSH module. Together, this data support an allosteric role for the CSH module in ACLY catalysis.
三磷酸腺苷柠檬酸裂解酶 (ACLY) 是胞浆核中乙酰辅酶 A 的主要来源，在许多疾病中存在异常调节，使其成为有吸引力的治疗靶点。ACLY 的结构研究揭示了一个中央同源四聚体柠檬酸合酶同源 (CSH) 模块，两侧为酰基辅酶 A 合成酶同源 (ASH) 结构域，ATP 和柠檬酸结合 ASH 结构域，而 CoA 结合 ASH-CSH 界面以产生乙酰辅酶 A 和草酰乙酸产物。CSH 模块的特定催化作用及其内部包含的一个必需的 D1026A 残基一直存在争议。在这里，我们报告了 ACLY-D1026A 突变体的生化和结构分析，证明该突变体在 ASH 结构域中捕获了一个（3S）-柠檬酸酰基辅酶 A 中间体，其构象与乙酰辅酶 A 的形成不兼容，能够在 ASH 结构域中将乙酰辅酶 A 和 OAA 转化为（3S）-柠檬酸酰基辅酶 A，并且能够在 CSH 结构域中加载 CoA 和卸载乙酰辅酶 A。总的来说，这些数据支持 CSH 模块在 ACLY 催化中的变构作用。
