Department of Chemistry and Biochemistry , University of Arizona , Tucson , Arizona 85721 , United States.
J Am Chem Soc. 2019 Jul 3;141(26):10431-10439. doi: 10.1021/jacs.9b04515. Epub 2019 Jun 24.
The design of artificial enzymes is an emerging field of research. Although progress has been made, the catalytic proficiency of many designed enzymes is low compared to natural enzymes. Nevertheless, recently Hilvert et al. ( Nat. Chem. 2017, 9, 50-56) created a series of five artificial retro-aldolase enzymes via directed evolution, with the final variant exhibiting a rate comparable to the naturally occurring enzyme fructose 1,6 bisphosphate aldolase. We present a study of this system in atomistic detail that elucidates the effects of mutational changes on the chemical step. Transition path sampling is used to create ensembles of reactive trajectories, and committor analysis is used to identify the stochastic separatrix of each ensemble. The application of committor distribution analysis to constrained trajectories allows the identification of changes in important protein motions coupled to reaction across the generated series of the artificial retro-aldolases. We observed two different reaction mechanisms and analyzed the role of the residues participating in the reaction coordinate of each enzyme. However, only in the most evolved variant we identified a fast motion that promotes catalysis, suggesting that this rate promoting vibration was introduced during directed evolution. This study provides further evidence that protein dynamics must be taken into account in designing efficient artificial enzymes.
人工酶的设计是一个新兴的研究领域。尽管已经取得了进展，但许多设计的酶的催化效率仍然低于天然酶。然而，最近 Hilvert 等人（Nat. Chem. 2017, 9, 50-56）通过定向进化创造了一系列五种人工 retro-aldolase 酶，最终变体的速率可与天然存在的酶果糖 1,6 二磷酸醛缩酶相媲美。我们以原子细节呈现了对该系统的研究，阐明了突变变化对化学步骤的影响。过渡路径采样用于创建反应轨迹的集合，而配分分析用于识别每个集合的随机分隔线。将配分分布分析应用于受约束的轨迹可用于识别与整个人工 retro-aldolase 系列生成相关的重要蛋白质运动的变化。我们观察到两种不同的反应机制，并分析了参与每个酶反应坐标的残基的作用。然而，只有在最进化的变体中，我们才确定了一种促进催化的快速运动，这表明这种促进催化的振动是在定向进化过程中引入的。这项研究进一步证明，在设计高效人工酶时必须考虑蛋白质动力学。
