Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.
Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.
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硫酸酯酶的进化再利用：一种新的米氏复合物导致有效的过渡态电荷抵消。
DOI：
10.1073/pnas.1607817115
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发表时间：
2018
影响因子：
11.1
通讯作者：
Miton CM
中科院分区：
文献类型：
--
作者：
Miton CM
混杂酶的招募和进化优化是生物体快速适应不断变化的环境的关键。然而，我们对酶再利用的精确机制的理解是有限的：哪些活性位点特征能够实现对具有不同催化要求的多种底物的分子识别？为了深入了解混杂酶适应的分子决定因素，我们对芳基硫酸酯酶进行了实验室进化，以改善其最初较弱的苯基膦酸水解酶活性。进化轨迹导致苯基膦酸酯水解增强了 100,000 倍，而天然硫酸盐和混杂的磷酸单酯和二酯水解仅受到轻微影响（≤50 倍）。进化中间体的结构、动力学和计算机表征表明，两个关键突变 T50A 和 M72V 局部重塑了活性位点，改善了膦酸酯催化机制的可及性。沿轨迹测得的过渡态 (TS) 电荷变化表明形成了新的米氏复合体（E•S，酶-底物），并且 TS 中混杂膦酸酯的离去基团稳定性增强（β 离去基团从 -1.08 到 -0.42）。进化再利用不是改变催化机制，而是通过微调米氏复合体中膦酸的分子识别来实现，进而延伸到 TS 中。这种分子情景构成了仅基于酶灵活性和构象选择的适应的机械替代方案。相反，不同化学反应之间的快速功能转变依赖于允许多种底物结合模式的许可活性位点结构的高反应性。
The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme–substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (βleavinggroupfrom −1.08 to −0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.