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

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 (T_m) of 65 °C, 17 °C higher than that of the wild-type enzyme (TMO-WT). At 50 °C, the stabilities (t_1/2) 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.