Extreme Synergistic Mutational Effects in the Directed Evolution of a Baeyer–Villiger Monooxygenase as Catalyst for Asymmetric Sulfoxidation,Journal of the American Chemical Society

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Extreme Synergistic Mutational Effects in the Directed Evolution of a Baeyer–Villiger Monooxygenase as Catalyst for Asymmetric Sulfoxidation
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2014-11-24 , DOI: 10.1021/ja5098034
Zhi-Gang Zhang _{1,

2} , Richard Lonsdale _{1,

2} , Joaquin Sanchis ₃ , Manfred T. Reetz _{1,

2}

Affiliation

Structure-based directed evolution utilizing iterative saturation mutagenesis (ISM) has been applied to phenyl acetone monooxygenase (PAMO), a thermally robust Baeyer-Villiger monooxygenase, in the quest to access a mutant which displays reversed enantioselectivity in the asymmetric sulfoxidation of prochiral thioethers. Whereas WT PAMO leads to 90% ee in the sulfoxidation of p-methylbenzyl methyl thioether with preference for the (S)-sulfoxide, the evolved mutant I67Q/P440F/A442N/L443I is 95% (R)-selective in the reaction of this and other thioethers. Partial deconvolution of the (R)-selective mutant with generation of the respective four single mutants shows that all of them are (S)-selective, which points to pronounced synergism (cooperative nonadditivity) when they interact in concert. Complete deconvolution with formation of all combinatorial forms of the respective double and triple mutants allows the designed construction of a fitness landscape featuring all 24 upward pathways leading from WT to the (R)-selective quadruple mutant. In all 24 trajectories strong cooperative mutational effects were found as well, which indicates that such mutational changes in enzymes constitute nonlinear systems. A theoretical analysis based on induced fit docking explains many of the observed effects on a molecular level.

中文翻译：

Baeyer-Villiger 单加氧酶定向进化中的极端协同突变效应作为不对称亚硫酸化的催化剂

利用迭代饱和诱变 (ISM) 的基于结构的定向进化已应用于苯基丙酮单加氧酶 (PAMO)，这是一种耐热的拜尔-维利格单加氧酶，以寻求获得一种突变体，该突变体在前手性硫醚的不对称亚砜化中显示出反向对映选择性。鉴于 WT PAMO 在对甲基苄基甲基硫醚的亚砜氧化中产生 90% ee，优先选择 (S)-亚砜，而进化的突变体 I67Q/P440F/A442N/L443I 在该反应中具有 95% (R)-选择性和其他硫醚。(R)-选择性突变体的部分解卷积与相应四个单突变体的产生表明它们都是 (S)-选择性的，这表明当它们协同相互作用时有明显的协同作用（合作非可加性）。通过形成相应双突变体和三重突变体的所有组合形式的完全解卷积，可以设计出具有从 WT 到 (R) 选择性四重突变体的所有 24 条向上途径的适应性景观。在所有 24 条轨迹中也发现了强烈的协同突变效应，这表明酶的这种突变变化构成了非线性系统。基于诱导拟合对接的理论分析解释了许多在分子水平上观察到的影响。这表明酶的这种突变变化构成了非线性系统。基于诱导拟合对接的理论分析在分子水平上解释了许多观察到的影响。这表明酶的这种突变变化构成了非线性系统。基于诱导拟合对接的理论分析解释了许多在分子水平上观察到的影响。

更新日期：2014-11-24

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