Counteracting the Activity-Diastereoselectivity Trade-Off of l-Threonine Aldolase by Regulating the Proton Transfer Microenvironment,Advanced Synthesis & Catalysis

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Counteracting the Activity-Diastereoselectivity Trade-Off of l-Threonine Aldolase by Regulating the Proton Transfer Microenvironment
Advanced Synthesis & Catalysis ( IF 4.4 ) Pub Date : 2022-11-19 , DOI: 10.1002/adsc.202201006
Sai Fang ₁ , Haoran Yu ₁ , Lanxin Xiao ₁ , Zhe Wang ₁ , Yixuan Lei ₁ , Gang Xu ₂ , Li-Rong Yang ₂ , Wenlong Zheng ₁ , Jian-Ping Wu ₂

Affiliation

l-threonine aldolase (LTA) is a vital tool for the production of β-hydroxy-α-amino acids, important pharmaceutical intermediates with two chiral centres. However, the trade-off between activity and diastereoselectivity seriously hinders the application of LTA. Here, microenvironment of the proton transfer was regulated to improve the enzyme activity while avoiding the loss of diastereoselectivity. A combinatorial active-site saturation test (CAST) strategy was applied to engineer the microenvironment of the key histidines H86 and H128 involved in proton transfer. Except for the amino acid residues tunning diastereoselectivity, a total of 18 (9+9) residues lining around H86 and H128 were investigated. As a result, two variants, RS1-T92V and RS1-E123R, were obtained with specific activity from 9.61 U/mg to 11.24 U/mg and 14.41 U/mg, respectively. By combinatorial mutagenesis, a double-point mutant RS1-VR (T92V/E123R) was obtained with specific activity reaching 18.65 U/mg that was two-fold of the original strain (RS1). Notably, the mutant RS1-VR remained a high de value of 94.21%. Molecular dynamics (MD) simulations provided insights into the mechanism of activity-diastereoselectivity trade-off. The improvement of microenvironment contributes to reduce the swing amplitude of the side chain of H86, resulting in the proton transfer more efficient. This work provides a strategy of regulating the proton transfer microenvironment for counteracting the trade-off between activity and diastereoselectivity in protein engineering.

中文翻译：

通过调节质子转移微环境抵消 l-苏氨酸醛缩酶的活性-非对映选择性权衡

l -苏氨酸醛缩酶 ( L TA) 是生产β -羟基- α的重要工具-氨基酸，具有两个手性中心的重要医药中间体。然而，活性和非对映选择性之间的权衡严重阻碍了LTA的应用。在这里，调节质子转移的微环境以提高酶活性，同时避免非对映选择性的损失。应用组合活性位点饱和测试 (CAST) 策略来设计参与质子转移的关键组氨酸 H86 和 H128 的微环境。除了调节非对映选择性的氨基酸残基外，还研究了排列在 H86 和 H128 周围的总共 18 (9+9) 个残基。结果，获得了两种变体，RS1-T92V 和 RS1-E123R，比活性分别为 9.61 U/mg 至 11.24 U/mg 和 14.41 U/mg。通过组合诱变，获得双点突变株RS1-VR（T92V/E123R），比活性达到18.65 U/mg，是原株（RS1）的2倍。值得注意的是，突变体 RS1-VR 保持高de值94.21%。分子动力学 (MD) 模拟提供了对活性-非对映选择性权衡机制的见解。微环境的改善有助于降低H86侧链的摆动幅度，从而提高质子转移效率。这项工作提供了一种调节质子转移微环境的策略，以抵消蛋白质工程中活性和非对映选择性之间的权衡。

更新日期：2022-11-19

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