Rational-design engineering to create the flavin reductase variants with thermostable and solvent-tolerant properties.

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 degrees C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 degrees 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.