Department of Physics, The Chinese University of Hong Kong, Shatin, Hong Kong.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America.
PLoS Comput Biol. 2018 Oct 26;14(10):e1006511. doi: 10.1371/journal.pcbi.1006511. eCollection 2018 Oct.
Substrate permissiveness has long been regarded as the raw materials for the evolution of new enzymatic functions. In land plants, hydroxycinnamoyltransferase (HCT) is an essential enzyme of the phenylpropanoid metabolism. Although essential enzymes are normally associated with high substrate specificity, HCT can utilize a variety of non-native substrates. To examine the structural and dynamic basis of substrate permissiveness in this enzyme, we report the crystal structure of HCT from Selaginella moellendorffii and molecular dynamics (MD) simulations performed on five orthologous HCTs from several major lineages of land plants. Through altogether 17-μs MD simulations, we demonstrate the prevalent swing motion of an arginine handle on a submicrosecond timescale across all five HCTs, which plays a key role in native substrate recognition by these intrinsically promiscuous enzymes. Our simulations further reveal how a non-native substrate of HCT engages a binding site different from that of the native substrate and diffuses to reach the catalytic center and its co-substrate. By numerically solving the Smoluchowski equation, we show that the presence of such an alternative binding site, even when it is distant from the catalytic center, always increases the reaction rate of a given substrate. However, this increase is only significant for enzyme-substrate reactions heavily influenced by diffusion. In these cases, binding non-native substrates 'off-center' provides an effective rationale to develop substrate permissiveness while maintaining the native functions of promiscuous enzymes.
长久以来，人们一直认为基质宽容性是新酶功能进化的原材料。在陆地植物中，羟基肉桂酰转移酶（HCT）是苯丙烷代谢中的一种必需酶。尽管必需酶通常与高底物特异性相关，但 HCT 可以利用多种非天然底物。为了研究该酶中底物宽容性的结构和动力学基础，我们报告了来自卷柏的 HCT 的晶体结构以及对来自陆地植物几个主要谱系的五个同源 HCT 进行的分子动力学（MD）模拟。通过总共 17 μs 的 MD 模拟，我们证明了在所有五个 HCT 中，一个精氨酸手柄在亚微秒时间尺度上普遍摆动的运动，该运动在这些固有混杂酶识别天然底物中起着关键作用。我们的模拟还进一步揭示了 HCT 的非天然底物如何与天然底物不同的结合位点结合并扩散到达催化中心及其共底物。通过数值求解 Smoluchowski 方程，我们表明即使替代结合位点远离催化中心，其始终会增加给定底物的反应速率。然而，对于受扩散影响较大的酶-底物反应，这种增加才具有重要意义。在这些情况下，“偏心”结合非天然底物为在保持混杂酶的天然功能的同时发展底物宽容性提供了有效的理论依据。
