Substrate-Dependent Mobile Loop Conformational Changes in Alkanesulfonate Monooxygenase from Accelerated Molecular Dynamics
Substrate-Dependent Mobile Loop Conformational Changes in Alkanesulfonate Monooxygenase from Accelerated Molecular Dynamics
复制标题
加速分子动力学中烷磺酸单加氧酶的底物依赖性移动环构象变化
DOI：
10.1021/acs.biochem.0c00633
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Acevedo, Orlando
中科院分区：
文献类型：
--
作者：
Thakur, Abhishek;Somai, Shruti;Yue, Kun;Ippolito, Nicole;Pagan, Dianne;Xiong, Jingyuan;Ellis, Holly R.;Acevedo, Orlando
底物诱导的链烷磺酸单加氧酶（SsuD）的构象变化是催化的关键，并导致动态环区域和活性位点之间的独特的相互作用。加速分子动力学（aMD）模拟已经进行了研究，以检查这种潜在的相关性，通过研究野生型SsuD和变体酶结合不同的组合还原黄素（FMNH 2），C4 a-过氧黄素中间体（FMNOO-），和辛烷磺酸盐（OCS）。鉴定了三种不同的移动的环构象：“开放”、“闭合”和“半闭合”。无衬底SsuD系统具有宽开口，能够为衬底提供进入活性位点的完全通路。在结合FMNH 2时，SsuD采用封闭构象，其将在不存在OCS的情况下防止非生产性氧化反应。两个盐桥，Asp 111-Arg 263和Glu 205-Arg 271，被确定为在维持闭合构象中特别重要。实验取代Arg 271丙氨酸没有改变的催化活性，但在还原黄素的存在下的变体更容易蛋白水解消化相比，野生型。当FMNH 2和OCS均以SsuD结合时，由于His 124和Phe 261之间存在良好的π-π相互作用，形成了第二种构象。因此，在稳态动力学测定中没有观察到F261 W SsuD变体的活性。这种半封闭的构象可能更适合于接受O2进入结合口袋和/或可以适当地定向活性位点，以进行氧分解裂解。最后，模拟SsuD同时绑定与FMNOO-和OCS发现一个开放的移动的环区域，这表明替代黄素中间体可能参与的反应机制。
Substrate-induced conformational changes present in alkanesulfonate monooxygenase (SsuD) are crucial to catalysis and lead to distinct interactions between a dynamic loop region and the active site. Accelerated molecular dynamics (aMD) simulations have been carried out to examine this potential correlation by studying wild-type SsuD and variant enzymes bound with different combinations of reduced flavin (FMNH2), C4a-peroxyflavin intermediate (FMNOO–), and octanesulfonate (OCS). Three distinct mobile loop conformations were identified: “open”, “closed”, and “semiclosed”. The substrate-free SsuD system possessed a wide opening capable of providing full access for substrates to enter the active site. Upon binding FMNH2, SsuD adopts a closed conformation that would prevent unproductive oxidation reactions in the absence of OCS. Two salt bridges, Asp111-Arg263 and Glu205-Arg271, were identified as particularly important in maintaining the closed conformation. Experimental substitution of Arg271 to Ala did not alter the catalytic activity, but the variant in the presence of reduced flavin was more susceptible to proteolytic digestion compared to wild-type. With both FMNH2and OCS bound in SsuD, a second conformation was formed dependent upon a favorable π–π interaction between His124 and Phe261. Accordingly, there was no observed activity with the F261W SsuD variant in steady-state kinetic assays. This semiclosed conformation may be more appropriate for accepting O2into the binding pocket and/or may properly orient the active site for the ensuing oxygenolytic cleavage. Finally, simulations of SsuD simultaneously bound with FMNOO–and OCS found an open mobile loop region that suggests alternative flavin intermediates may participate in the reaction mechanism.