Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation
Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation
复制标题
较短的烷磺酸盐碳链破坏了 SsuD 脱磺化活性位点结构的稳定性
DOI：
10.1021/acs.biochem.2c00586
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发表时间：
2023
期刊：
影响因子：
2.9
通讯作者：
Ellis, Holly R.
中科院分区：
文献类型：
--
作者：
Somai, Shruti;Yue, Kun;Acevedo, Orlando;Ellis, Holly R.
当硫含量有限时，细菌已经进化到利用替代的有机硫磺来源。硫源受限时表达的SSUE/SSUD和MsuE/MSUD酶负责向特定细菌提供烷基磺酸盐形式的硫。在这项研究中，我们评估了为什么在一些细菌中需要两种结构和功能相似的FMNH2依赖的单加氧酶(MSUD和SsuD)来获得烷基磺酸盐。在脱磺试验中，MSUD能够利用整个系列的烷基磺酸盐(C1-C10)。然而，SsuD不能利用较小的烷基磺酸盐底物。有趣的是，SsuD与甲烷磺酸(MES)的结合亲和力(15±1μM)与MSUD(12±1μM)相似，尽管SsuD不能催化MES底物的脱磺反应。SsuD和MSUD在含有MES和辛烷磺酸盐(OCS)的FMNH2存在时，蛋白降解敏感性降低。与SsuD相比，MSUD/FMNH2与MES和OCS的络合物在可比条件下观察到更紧密的环闭合。用加速分子动力学模拟对SsuD/FMNH2/MES结构进行了分析，发现MES有三种不同的构象，说明了结合结构的不稳定性。即使当MES与活性中心内的OCS以类似的方式结合时，较小的烷烃链也会导致FMNH2的位移，从而不再能够催化MES的脱磺反应。SsuD的活性中心需要较长的烷烃链来保持适当的脱磺构型。
Bacteria have evolved to utilize alternative organosulfur sources when sulfur is limiting. The SsuE/SsuD and MsuE/MsuD enzymes expressed when sulfur sources are restricted, are responsible for providing specific bacteria with sulfur in the form of alkanesulfonates. In this study, we evaluated why two structurally and functionally similar FMNH2-dependent monooxygenase enzymes (MsuD and SsuD) are needed for the acquisition of alkanesulfonates in some bacteria. In desulfonation assays, MsuD was able to utilize the entire range of alkanesulfonates (C1–C10). However, SsuD was not able to utilize smaller alkanesulfonate substrates. Interestingly, SsuD had a similar binding affinity for methanesulfonate (MES) (15 ± 1 μM) as MsuD (12 ± 1 μM) even though SsuD was not able to catalyze the desulfonation of the MES substrate. SsuD and MsuD showed decreased proteolytic susceptibility in the presence of FMNH2with MES and octanesulfonate (OCS). Tighter loop closure was observed for the MsuD/FMNH2complex with MES and OCS compared to SsuD under comparable conditions. Analysis of the SsuD/FMNH2/MES structure using accelerated molecular dynamics simulations found three different conformations for MES, demonstrating the instability of the bound structure. Even when MES was bound in a similar fashion to OCS within the active site, the smaller alkane chain resulted in a shift of FMNH2so that it was no longer in a position to catalyze the desulfonation of MES. The active site of SsuD requires a longer alkane chain to maintain the appropriate architecture for desulfonation.