Promiscuous Esterases Counterintuitively Are Less Flexible than Specific Ones

Understanding mechanisms of promiscuity is increasingly important from a fundamental and application point of view. As to enzyme structural dynamics, more promiscuous enzymes generally have been recognized to also be more flexible. However, examples for the opposite received much less attention. Here, we exploit comprehensive experimental information on the substrate promiscuity of 147 esterases tested against 96 esters together with computationally efficient rigidity analyses to understand the molecular origin of the observed promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly less flexible than specific ones, are significantly more thermostable, and have a significantly increased specific activity. These results may be reconciled with a model according to which structural flexibility in the case of specific esterases serves for conformational proofreading. Our results signify that an esterase sequence space can be screened by rigidity analyses for promiscuous esterases as starting points for further exploration in biotechnology and synthetic chemistry.从基础研究与应用视角出发，解析酶底物混杂性（promiscuity）的机制愈发重要。就酶的结构动态特性而言，学界普遍认为底物宽泛性更强的酶通常柔性也更高。然而，与此相悖的案例却鲜有受到关注。本研究依托针对147种酯酶在96种酯类底物下的底物混杂性所开展的系统性实验数据，结合计算高效的刚性分析方法，探究了观测到的底物混杂性差异的分子起源。出乎意料的是，我们的研究数据显示，具有底物混杂性的酯酶相较于专一性酯酶，其柔性显著更低、热稳定性显著更高，且比活显著提升。上述结果可通过下述模型得到合理解释：专一性酯酶的结构柔性用于构象校正。本研究表明，可通过刚性分析对酯酶序列空间进行筛选，获取具有底物混杂性的酯酶，将其作为生物技术与合成化学后续探索的起点。