Creating Flavin Reductase Variants with Thermostable and Solvent‐Tolerant Properties by Rational‐Design Engineering

Abstract

Abstract We have employed computational approaches—FireProt and FRESCO—to predict thermostable variants of the reductase component (C 1 ) of (4‐hydroxyphenyl)acetate 3‐hydroxylase. With the additional aid of experimental results, two C 1 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 1 variants remain active and generate reduced flavin mononucleotide (FMNH − ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C 2 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 1 enzyme can lead to broad‐spectrum uses of C 1 as a redox biocatalyst for future industrial applications.