Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series
Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series
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DOI：
10.1016/j.jmb.2009.12.031
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发表时间：
2010-03-05
影响因子：
5.6
通讯作者：
Tawfik, Dan S.
中科院分区：
文献类型：
--
作者：
Khersonsky, Olga;Rothlisberger, Daniela;Tawfik, Dan S.
通过分析天然酶来理解酶的催化作用是一个艰巨的挑战--它们的活性部位很复杂，结合了许多相互作用和精细协调的催化力。对更基本(Wo)人造酶的研究为更好地理解酶催化提供了一个独特的机会。KE07是一种通过计算设计的Kemp消除酶，它利用谷氨酸侧链作为关键质子提取步骤的催化基础，并通过非极性结合位点指导底物结合，通过七轮随机突变和选择对KE07进行了优化，使催化效率提高了200倍。在这里，我们详细地描述了定向进化过程以及设计的KE07及其进化变体的生物物理和结晶学研究。对KE07‘S的活性进行了优化，得到了与S(-1)M-1相似的k(CAT)/K-M值和接近10(6)倍的速率加速(k(CAT)/k(UNCAT))，涉及多达8个突变的掺入。这些突变导致进化的KE07的整体热力学稳定性及其活性部位的构象稳定性显著降低。我们发现突变对KE07的S活性提高有两个主要贡献：(I)引入了新的盐桥，以纠正原始设计中的一个错误，该设计将赖氨酸放置在离开基质子化的错误中，而没有考虑它与催化谷氨酸的“猝灭”相互作用；(Ii)环境的调节，催化碱基的pK(A)，以及它与底物的相互作用，通过活性中心周围几个带电残基组成的氢键网络的演变来实现。(C)2010爱思唯尔有限公司。保留所有权利。
Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge-their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k(cat)/K-M value of similar to 2600 s(-1) M-1 and an similar to 10(6)-fold rate acceleration (k(cat)/k(uncat)) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its "quenching" interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK(a) of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site. (C) 2010 Elsevier Ltd. All rights reserved.