Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation†

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY RSC Chemical Biology Pub Date : 2024-07-27 DOI:10.1039/D4CB00102H
Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat and Pimchai Chaiyen
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Abstract

Tryptophan 2-monooxygenase (TMO) is an FAD-bound flavoenzyme which catalyzes the oxidative decarboxylation of L-tryptophan to produce indole-3-acetamide (IAM) and carbon dioxide. The reaction of TMO is the first step of indole-3-acetic acid (IAA) biosynthesis. Although TMO is of interest for mechanistic studies and synthetic biology applications, the enzyme has low thermostability and soluble expression yield. Herein, we employed a combined approach of rational design using computational tools with site-saturation mutagenesis to screen for TMO variants with significantly improved thermostability properties and soluble protein expression. The engineered TMO variants, TMO-PWS and TMO-PWSNR, possess melting temperatures (Tm) of 65 °C, 17 °C higher than that of the wild-type enzyme (TMO-WT). At 50 °C, the stabilities (t1/2) of TMO-PWS and TMO-PWSNR were 85-fold and 92.4-fold higher, while their soluble expression yields were 1.4-fold and 2.1-fold greater than TMO-WT, respectively. Remarkably, the kinetic parameters of these variants were similar to those of the wild-type enzymes, illustrating that they are promising candidates for future studies. Molecular dynamic simulations of the wild-type and thermostable TMO variants identified key interactions for enhancing these improvements in the biophysical properties of the TMO variants. The introduced mutations contributed to hydrogen bond formation and an increase in the regional hydrophobicity, thereby, strengthening the TMO structure.

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通过半理性酶工程提高色氨酸 2-单加氧酶的热稳定性:尽量减少实验研究的战略设计
色氨酸 2-单氧化酶(TMO)是一种与 FAD 结合的黄酶,它催化 L-色氨酸氧化脱羧,生成吲哚-3-乙酰胺(IAM)和二氧化碳。TMO 反应是吲哚-3-乙酸(IAA)生物合成的第一步。虽然 TMO 对机理研究和合成生物学应用很有意义,但该酶的热稳定性和可溶性表达量都很低。在此,我们采用了一种利用计算工具进行合理设计与定点饱和诱变相结合的方法,筛选出热稳定性和可溶性蛋白表达量均有显著改善的 TMO 变体。所设计的 TMO 变体 TMO-PWS 和 TMO-PWSNR 的熔化温度(Tm)为 65 ˚C,比野生型酶(TMO-WT)的熔化温度高 17 ˚C。在 50 ˚C 时,TMO-PWS 和 TMO-PWSNR 的稳定性(t1/2)分别比 TMO-WT 高 85 倍和 92.4 倍,可溶性表达量分别比 TMO-WT 高 1.4 倍和 2.1 倍。值得注意的是,这些变体的动力学参数与野生型酶相似,这说明它们是未来研究的候选对象。对野生型和恒温 TMO 变体的分子动力学模拟确定了关键的相互作用,这些相互作用提高了 TMO 变体的生物物理特性。引入的突变有助于氢键的形成和区域疏水性的增加,从而加强了 TMO 的结构。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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