Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

中文翻译：

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致病真菌乙酰辅酶A合成酶的反应和底物特异性机制的结构表征

乙酰辅酶 A 合成酶 (ACS) 是A cyl-CoA/ N RPS/ L荧光素酶 (ANL) 超家族酶，可将乙酸盐与 CoA 结合以生成乙酰 CoA，乙酰 CoA 是真核生物和原核生物中中心碳代谢的关键组成部分。正常哺乳动物细胞不依赖 ACS，而肿瘤细胞、真菌和寄生虫则依赖乙酸作为乙酰辅酶 A 的前体。因此，ACS 已成为潜在的药物靶点。作为开发抗真菌 ACS 抑制剂计划的一部分，我们使用生化和结构研究对来自五种不同人类真菌病原体的真菌 ACS 进行了表征。ACSs 催化一个两步反应，包括乙酸腺苷酸化和 CoA 硫酯化。我们的结构研究捕获了这两个半反应的每个步骤，包括第一个半反应的乙酰腺苷酸中间体在腺苷酸化构象和硫酯化构象中，从而提供了反应机理的详细图片。我们还使用了一系列越来越大的烷基腺苷酯作为化学探针来表征乙酸盐在较大羧酸底物上的精细 ACS 特异性的结构基础。与之前其他酶的生化和遗传数据一致，与这些探针结合的真菌 ACS 的结构表明，一个关键的色氨酸残基限制了烷基结合位点的大小，并迫使较大的烷基链采用高能构象异构体，不利于它们的有效结合。一起，