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

• Published in: Chembiochem : a European journal of chemical biology Vol. 21; no. 10; pp. 1481 - 1491
• Main Authors: Maenpuen, Somchart, Pongsupasa, Vinutsada, Pensook, Wiranee, Anuwan, Piyanuch, Kraivisitkul, Napatsorn, Pinthong, Chatchadaporn, Phonbuppha, Jittima, Luanloet, Thikumporn, Wijma, Hein J., Fraaije, Marco W., Lawan, Narin, Chaiyen, Pimchai, Wongnate, Thanyaporn
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 15.05.2020
• Subjects: Acetic acid; biocatalysis; Chemical reactions; computational chemistry; Computer applications; Computer simulation; Design engineering; Flavin mononucleotide; Flavin reductase; flavoproteins; Heat treatment; Heat treatments; Hydrophobicity; Hydroxylase; Industrial applications; Molecular dynamics; Monooxygenase; Mutation; Reductases; Solvents; Temperature tolerance; Thermal stability; thermostable enzymes; computational chemistry; reductases; biocatalysis; flavoproteins; thermostable enzymes
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.201900737

We have employed computational approaches—FireProt and FRESCO—to predict thermostable variants of the reductase component (C1) of (4‐hydroxyphenyl)acetate 3‐hydroxylase. With the additional aid of experimental results, two C1 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 C1 variants remain active and generate reduced flavin mononucleotide (FMNH−) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 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 C1 enzyme can lead to broad‐spectrum uses of C1 as a redox biocatalyst for future industrial applications. Some like it hot: Two computational approaches were used to predict thermostable variants of the reductase C1. The A166L and A58P thermotolerant variants showed improvements of 2.6–5.6 °C in thermostability and of two‐ to 3.5‐fold in catalytic efficiency, as well as solvent tolerance. These thermostable C1 variants should in the future be useful industrial biocatalysts for performing redox reactions.