Wójcik-Augustyn Anna, Johansson A Johannes, Borowski Tomasz
Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Cracow, Poland.
Swedish Nuclear Fuel and Waste Management Co (SKB), Box 3091, 169 03 Solna, Sweden.
Comput Struct Biotechnol J. 2019 Jun 18;17:770-784. doi: 10.1016/j.csbj.2019.06.016. eCollection 2019.
ATPS Sulfurylase (ATPS) is the first of three enzymes in the sulfate reduction pathway - one of the oldest metabolic pathways on Earth, utilized by Sulfate Reducing Bacteria (SRB). Due to the low redox potential of the sulfate ion, its reduction requires activation via formation of adenosine 5'-phosphosulfate (APS), which is catalyzed by ATPS. Dispersion-corrected hybrid density functional theory (DFT/B3LYP-D3) was used to test three reaction mechanisms proposed for conversion of ATP to APS: two-step SN-1 reaction running through AMP anhydride intermediate, two-step reaction involving cyclic AMP intermediate and direct SN-2 conversion of ATP to APS molecule. The study employed five different cluster models of the ATPS active site: one containing magnesium cation and four without it, constructed based on the crystal structure (PDB code: 1G8H) solved for ATPS from in complex with APS and pyrophosphate (PPi), where Mg was not detected. The model with magnesium ion was constructed based on the representative structure obtained from trajectory analysis of the molecular dynamics simulations (MD) performed for the hexameric ATPS-APS-Mg-PPi complex. The results obtained for all considered models suggest that ATPS-AMP anhydride intermediate is a highly energetic and unstable complex, while formation of cyclic AMP molecule requires formation of unfavorable hypervalent geometry at the transition state. Among all tested mechanism, the energetically most feasible mechanism of the ATPS reaction is SN-2 one-step conversion of ATP to APS occurring via a pentavalent transition state. Interestingly, such a reaction is inhibited by the presence of Mg in the ATPS active site. Magnesium cation forces unfavorable geometry of reactants for SN-2 mechanism and formation of pentavalent transition state. Such a reaction requires rearrangement of Mg ligands, which raises the barrier from 11-14 kcal/mol for the models without Mg to 48 kcal/mol for model with magnesium ion included.
腺苷 5'-磷酸硫酸化酶（ATPS）是硫酸盐还原途径中三种酶的第一种，硫酸盐还原途径是地球上最古老的代谢途径之一，被硫酸盐还原细菌（SRB）所利用。由于硫酸根离子的氧化还原电位较低，其还原需要通过形成腺苷 5'-磷酸硫酸（APS）来激活，这一过程由 ATPS 催化。采用色散校正的杂化密度泛函理论（DFT/B3LYP-D3）来测试提出的三种将 ATP 转化为 APS 的反应机制：通过 AMP 酐中间体的两步 SN-1 反应、涉及环 AMP 中间体的两步反应以及 ATP 直接 SN-2 转化为 APS 分子。该研究采用了 ATPS 活性位点的五种不同簇模型：一种含有镁阳离子，四种不含镁阳离子，这些模型基于与 APS 和焦磷酸（PPi）形成复合物的 ATPS 的晶体结构（PDB 代码：1G8H）构建，其中未检测到镁。含镁离子的模型基于对六聚体 ATPS-APS-Mg-PPi 复合物进行分子动力学模拟（MD）的轨迹分析获得的代表性结构构建。所有考虑模型的结果表明，ATPS-AMP 酐中间体是一种高能且不稳定的复合物，而环 AMP 分子的形成需要在过渡态形成不利的高价几何结构。在所有测试机制中，ATPS 反应在能量上最可行的机制是通过五价过渡态将 ATP 直接 SN-2 一步转化为 APS。有趣的是，ATPS 活性位点中镁的存在会抑制这种反应。镁阳离子迫使反应物形成不利于 SN-2 机制和五价过渡态形成的几何结构。这种反应需要重新排列镁配体，这使得不含镁的模型的能垒从 11 - 14 kcal/mol 增加到包含镁离子的模型的 48 kcal/mol。
