Laboratory of Organic Chemistry, ETH Zürich , 8093 Zürich, Switzerland.
J Am Chem Soc. 2017 Sep 13;139(36):12541-12549. doi: 10.1021/jacs.7b05796. Epub 2017 Aug 28.
De novo biocatalysts have been successfully generated by computational design and subsequent experimental optimization. Here, we examined the evolutionary history of the computationally designed (retro-)aldolase RA95. The modest activity of the starting enzyme was previously improved 10-fold over many rounds of mutagenesis and screening to afford a proficient biocatalyst for enantioselective cleavage and synthesis of β-hydroxyketones. Using a set of representative RA95 variants, we probed individual steps in the multistep reaction pathway to determine which processes limit steady-state turnover and how mutations that accumulated along the evolutionary trajectory influenced the kinetic mechanism. We found that the overall rate-limiting step for aldol cleavage shifted from C-C bond scission (or an earlier step in the pathway) for the computational design to product release for the evolved enzymes. Specifically, interconversion of Schiff base and enamine intermediates, formed covalently between acetone and the catalytic lysine residue, was found to be the slowest step for the most active variants. A complex hydrogen bond network of four active site residues, which was installed in the late stages of laboratory evolution, apparently enhances lysine reactivity and facilitates efficient proton shuffling. This catalytic tetrad accounts for the tremendous rate acceleration observed for all steps of the mechanism, most notably Schiff base formation and hydrolysis. Comparison of our results with kinetic and structural studies on natural aldolases provides valuable feedback for computational enzyme design and laboratory evolution approaches alike.
通过计算设计和后续的实验优化，已经成功地产生了从头生物催化剂。在这里，我们研究了经过计算设计（回溯）醛缩酶 RA95 的进化历史。该起始酶的活性适中，经过多轮诱变和筛选，其活性提高了 10 倍，从而成为用于对映选择性断裂和合成β-羟基酮的高效生物催化剂。使用一组代表性的 RA95 变体，我们探测了多步反应途径中的各个步骤，以确定哪些过程限制了稳态周转率，以及沿着进化轨迹积累的突变如何影响动力学机制。我们发现，对于醛裂解，从计算设计的 C-C 键断裂（或途径中的早期步骤）到进化酶的产物释放，整体限速步骤发生了转变。具体来说，在最活跃的变体中，形成于丙酮和催化赖氨酸残基之间的席夫碱和烯胺中间体的互变被发现是最慢的步骤。四个活性位点残基的复杂氢键网络，在实验室进化的后期阶段安装，显然增强了赖氨酸的反应性并促进了有效的质子转移。该催化四联体解释了对机制的所有步骤（尤其是席夫碱形成和水解）观察到的巨大速率加速。将我们的结果与天然醛缩酶的动力学和结构研究进行比较，为计算酶设计和实验室进化方法提供了有价值的反馈。
