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Exploring the Catalytic Mechanism of Alkanesulfonate Monooxygenase Using Molecular Dynamics

机译：用分子动力学探索链烷磺酸盐单氧化酶的催化机制

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摘要

The complex mechanistic properties of alkanesulfonate monooxygenase (SsuD) provide a particular challenge for identifying catalytically relevant amino acids. In response, a joint computational and experimental study was conducted to further elucidate the SsuD mechanism. Extensive unbiased molecular dynamics (MD) simulations were performed for six SsuD systems: (1) substrate-free, (2) bound with FMNH2, (3) bound with a C4a-peroxyflavin intermediate (FMNOO~?), (4) bound with octanesulfonate (OCS), (5) co-bound with FMNH_2 and OCS, and (6) co-bound with FMNOO? and OCS. A previous theoretical study suggested that salt bridges between Arg297 and Glu20 or Asp111 initiated conformational changes critical for catalysis. However, our MD simulations and steady-state kinetic experiments did not corroborate this result. Similar kcat/Km values for both the E20A and D111A SsuD variants to wild-type SsuD suggest that the salt bridges are not critical to the desulfonation mechanism. Instead, the predicted role of Arg297 is to favorably interact with the phosphate group of the reduced flavin. Concomitantly, Arg226 functioned as a “protection” group shielding FMNOO? from bulk solvent and was more pronounced when both FMNOO~? and OCS were bound. The stabilization of FMNOO? through electrostatic interactions with Arg226 would properly position the C4a peroxy group for the proposed nucleophilic attack on the sulfur of octanesulfonate.
机译：链烷磺酸盐单氧基酶（SSUD）的复杂机制性质为鉴定鉴定催化相关的氨基酸提供了特殊的挑战。作为响应，进行了联合计算和实验研究以进一步阐明SSUD机制。对六个SSUD系统进行广泛的无偏见分子动力学（MD）模拟：（1）无底底，（2）与FMNH2，（3）结合，与C4A-过氧吡喃蛋白中间体（FMNOO〜2）结合，（4）结合辛烷磺酸盐（OCS），（5）与FMNH_2和OCS共束，（6）与FMNOO共束吗？和ocs。以前的理论研究表明，arg297和glu20或asp111之间的盐桥发起了对催化关键的构象变化。但是，我们的MD模拟和稳态动力学实验并未证实了这一结果。对于野生型SSUD的E20A和D111A SSUD变体的类似KCAT / KM值表明盐桥对脱硫机制并不重要。相反，Arg297的预测作用是有利地与减少的黄素的磷酸盐基团相互作用。兼顾，arg226作为“保护”组屏蔽FMNOO？来自批量溶剂，在fmnoo〜？和oc是约束的。 FMNOO的稳定性？通过与Arg226的静电相互作用将适当地定位C4a过氧基团的辛磺酸硫磺所提出的亲核攻击。

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  • 来源
    《Biochemistry》 |2014年第20期|共10页
  • 作者

    Kira Armacost; Jonathan Musila; Symon Gathiaka; Holly R. Ellis; Orlando Acevedo;

  • 作者单位

    Department of Chemistry and Biochemistry Auburn University Auburn Alabama 36849 United States;

    Department of Chemistry and Biochemistry Auburn University Auburn Alabama 36849 United States;

    Department of Chemistry and Biochemistry Auburn University Auburn Alabama 36849 United States;

    Department of Chemistry and Biochemistry Auburn University Auburn Alabama 36849 United States;

    Department of Chemistry and Biochemistry Auburn University Auburn Alabama 36849 United States;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    Exploring; Monooxygenase; Dynamics;

    机译：探索;单氧基酶;动态;

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