Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand.
Molecular Medicine Research Group (MMRG), Division of Medical Technology, Faculty of Allied Health Sciences, Burapha University, Chonburi, 20131, Thailand.
Arch Biochem Biophys. 2020 Nov 15;694:108615. doi: 10.1016/j.abb.2020.108615. Epub 2020 Oct 2.
We previously demonstrated that the 130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was acylated at Lys and thus activated its hemolytic activity. Here, attempts were made to provide greater insights into such toxin activation via fatty-acyl modification by CyaC-acyltransferase. Non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaC were separately expressed in E. coli and subsequently purified by FPLC to near homogeneity. When effects of acyl-chain length were comparatively evaluated through CyaC-esterolysis using various p-nitrophenyl (pNP) derivatives, Michaelis-Menten steady-state kinetic parameters (K and k) of CyaC-acyltransferase revealed a marked preference for myristoyl (C) and palmitoyl (C) substrates of which catalytic efficiencies (k/K) were roughly the same (1.5 × 10 smM). However, pNP-palmitate (pNPP) gave the highest hemolytic activity of NA/CyaA-Hly after being acylated in vitro with a range of acyl-donor substrates. LC-MS/MS analysis confirmed such CyaC-mediated palmitoylation of CyaA-Hly occurring at Lys, denoting no requirement of an acyl carrier protein (ACP). A homology-based CyaC structure inferred a role of a potential catalytic dyad of conserved Ser and His residues in substrate esterolysis. CyaC-ligand binding analysis via molecular docking corroborated high-affinity binding of palmitate with its carboxyl group oriented toward such a dyad. Ala-substitutions of each residue (S30A or H33A) caused a drastic decrease in k/K of CyaC toward pNPP, and hence its catalytic malfunction through palmitoylation-dependent activation of NA/CyaA-Hly. Altogether, our present data evidently provide such preferential palmitoylation of CyaA-Hly by CyaC-acyltransferase through the enzyme Ser-His nucleophile-activation dyad in esterolysis of palmitoyl-donor substrate, particularly devoid of a natural acyl-ACP donor.
我们之前已经证明，在大肠杆菌中与 CyaC 酰基转移酶共同表达的百日咳博德特氏菌的约 130kDa CyaA-溶血素结构域（CyaA-Hly）在赖氨酸处被酰化，从而激活其溶血活性。在这里，我们试图通过 CyaC 酰基转移酶的脂肪酸酰基修饰来提供对这种毒素激活的更深入了解。非酰化的 CyaA-Hly（NA/CyaA-Hly）和 CyaC 分别在大肠杆菌中表达，并通过 FPLC 进一步纯化至近乎均一。当通过 CyaC-胆固醇酯酶解用各种对硝基苯酚（pNP）衍生物比较评估酰基链长度的影响时，CyaC 酰基转移酶的米氏稳态动力学参数（K 和 k）显示出对豆蔻酰（C）和棕榈酰（C）底物的明显偏好，其催化效率（k/K）大致相同（约 1.5×10 smM）。然而，在用一系列酰基供体底物体外酰化后，pNP-棕榈酸（pNPP）赋予 NA/CyaA-Hly 最高的溶血活性。LC-MS/MS 分析证实了 CyaC 介导的 CyaA-Hly 在赖氨酸处的棕榈酰化，这表明不需要酰基载体蛋白（ACP）。基于同源性的 CyaC 结构推断了保守的丝氨酸和组氨酸残基在底物酯解中的潜在催化二联体的作用。通过分子对接进行的 CyaC-配体结合分析证实了棕榈酸与二联体的羧基基团的高亲和力结合。每个残基（S30A 或 H33A）的 Ala 取代导致 CyaC 对 pNPP 的 k/K 急剧下降，从而通过依赖于棕榈酰化的 NA/CyaA-Hly 激活导致其催化功能失调。总之，我们目前的数据清楚地表明，CyaC 酰基转移酶通过酶丝氨酸-组氨酸亲核体激活二联体在棕榈酰供体底物的酯解中对 CyaA-Hly 进行优先棕榈酰化，特别是在没有天然酰基-ACP 供体的情况下。
