Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase.
Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase.
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DOI：
10.1021/jacs.0c10701
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发表时间：
2020-11-25
影响因子：
15
通讯作者：
Kamerlin SCL
中科院分区：
文献类型：
--
作者：
Biler M;Crean RM;Schweiger AK;Kourist R;Kamerlin SCL
细菌芳基丙二酸脱羧酶(AMDase)及其衍生突变体已成为获得多种高光学纯度手性芳香酸对映体的有效工具。然而，到目前为止，对这种酶的底物范围、活性和选择性的分子原理还知之甚少，这极大地阻碍了针对特定应用的改进酶变体的可预测性和设计。在这项工作中，对野生型AMDase及其变体进行了经验价键和元动力学模拟，以更好地理解决定反应结果的潜在分子过程。我们的结果清楚地再现了实验观察到的底物范围，并支持一种由酶切割的羧酸基的基态失稳驱动的机制。此外，我们的结果表明，在本工作中研究的未转化或转化较差的底物的情况下，这些底物结合时增加活性部位的溶剂暴露可能会扰乱负责促进AMDase催化的二氧化碳裂解的脆弱的相互作用网络。最后，我们的结果表明，在所研究的所有底物中，AMDase的CLG-IPL变体中的Pro-(R)优先切割为Pro-(S)羧酸基。这似乎是由于插入了六个氨基酸取代而产生的新的疏水口袋，亲(S)羧酸盐与之结合。我们的结果使我们能够深入了解决定AMDase选择性的紧密相互作用网络，这反过来为未来酶工程的靶标残基的鉴定提供了指导。
Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity, and selectivity of this enzyme are only poorly understood to date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metadynamics simulations were performed on wild-type AMDase and variants thereof to obtain a better understanding of the underlying molecular processes determining reaction outcome. Our results clearly reproduce the experimentally observed substrate scope and support a mechanism driven by ground-state destabilization of the carboxylate group being cleaved by the enzyme. In addition, our results indicate that, in the case of the nonconverted or poorly converted substrates studied in this work, increased solvent exposure of the active site upon binding of these substrates can disturb the vulnerable network of interactions responsible for facilitating the AMDase-catalyzed cleavage of CO2. Finally, our results indicate a switch from preferential cleavage of the pro-(R) to the pro-(S) carboxylate group in the CLG-IPL variant of AMDase for all substrates studied. This appears to be due to the emergence of a new hydrophobic pocket generated by the insertion of the six amino acid substitutions, into which the pro-(S) carboxylate binds. Our results allow insight into the tight interaction network determining AMDase selectivity, which in turn provides guidance for the identification of target residues for future enzyme engineering.
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影响因子：
2.9
作者：
Feng, WY;Austin, TJ;Wu, WM
通讯作者：
Wu, WM
影响因子：
5.2
作者：
Assmann, Miriam;Muegge, Carolin;Kara, Selin
通讯作者：
Kara, Selin
影响因子：
3.2
作者：
Gassmeyer, Sarah Katharina;Yoshikawa, Hiroyuki;Kourist, Robert
通讯作者：
Kourist, Robert
影响因子：
3.3
作者：
Boresch, S;Tettinger, F;Karplus, M
通讯作者：
Karplus, M
影响因子：
46.9
作者：
通讯作者：
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