Department of Biochemistry, Faculty of Science, Burapha University, 169 Long-Hard Bangsaen Road, Chonburi, 20131, Thailand.
School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), 555 Moo 1 Payupnai, Wangchan, Rayong, 21210, Thailand.
Chembiochem. 2020 May 15;21(10):1481-1491. doi: 10.1002/cbic.201900737. Epub 2020 Feb 21.
We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C variants remain active and generate reduced flavin mononucleotide (FMNH ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C enzyme can lead to broad-spectrum uses of C as a redox biocatalyst for future industrial applications.
我们采用了计算方法——FireProt 和 FRESCO——来预测（4-羟基苯基）乙酸 3-羟化酶还原酶成分（C）的热稳定变体。在实验结果的额外帮助下，确定了两个 C 变体，A166L 和 A58P，为耐热酶，热稳定性提高了 2.6-5.6°C，催化效率提高了 2-3.5 倍。在 45°C 下热处理后，两种热稳定的 C 变体仍然保持活性，并比野生型（WT）更有效地生成还原型黄素单核苷酸（FMNH），用于细菌荧光素和单加氧酶 C 催化的反应。除了耐热性外，A166L 和 A58P 变体还表现出溶剂耐受性。300-500 K 的分子动力学（MD）模拟（6 ns）表明，A166 突变为 L 和 A58 突变为 P 导致结构变化，增加了疏水性相互作用的稳定性，从而提高了热稳定性。我们的研究结果表明，C 酶的热稳定性提高可以使其作为氧化还原生物催化剂在未来的工业应用中得到广泛应用。
