Galmés Miquel À, Nödling Alexander R, Luk Louis, Świderek Katarzyna, Moliner Vicent
Institute of Advanced Materials (INAM), Universitat Jaume I, 12071 Castellón, Spain.
Cardiff Catalysis Institute, School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom.
ACS Catal. 2021 Jul 16;11(14):8635-8644. doi: 10.1021/acscatal.1c02150. Epub 2021 Jun 30.
Convergent evolution has resulted in nonhomologous enzymes that contain similar active sites that catalyze the same primary and secondary reactions. Comparing how these enzymes achieve their reaction promiscuity can yield valuable insights to develop functions from the optimization of latent activities. In this work, we have focused on the promiscuous amidase activity in the esterase from (Bs2) and compared with the same activity in the promiscuous lipase B from (CALB). The study, combining multiscale quantum mechanics/molecular mechanics (QM/MM) simulations, deep machine learning approaches, and experimental characterization of Bs2 kinetics, confirms the amidase activity of Bs2 and CALB. The computational results indicate that both enzymes offer a slightly different reaction environment reflected by electrostatic effects within the active site, thus resulting in a different reaction mechanism during the acylation step. A convolutional neural network (CNN) has been used to understand the conserved amino acids among the evolved protein family and suggest that Bs2 provides a more robust protein scaffold to perform future mutagenesis studies. Results derived from this work will help reveal the origin of enzyme promiscuity, which will find applications in enzyme (re)design, particularly in creating a highly active amidase.
趋同进化产生了非同源酶，这些酶含有相似的活性位点，可催化相同的一级和二级反应。比较这些酶如何实现其反应多特异性，可为从潜在活性的优化中开发功能提供有价值的见解。在这项工作中，我们专注于来自嗜热栖热菌（Bs2）的酯酶中的多特异性酰胺酶活性，并与来自南极假丝酵母（CALB）的多特异性脂肪酶B中的相同活性进行比较。该研究结合了多尺度量子力学/分子力学（QM/MM）模拟、深度机器学习方法以及Bs2动力学的实验表征，证实了Bs2和CALB的酰胺酶活性。计算结果表明，两种酶在活性位点内的静电效应所反映的反应环境略有不同，从而在酰化步骤中导致不同的反应机制。卷积神经网络（CNN）已被用于了解进化后的蛋白质家族中的保守氨基酸，并表明Bs2为未来的诱变研究提供了更强大的蛋白质支架。这项工作的结果将有助于揭示酶多特异性的起源，这将在酶（重新）设计中找到应用，特别是在创建高活性酰胺酶方面。
