1c:I[776865,["/_next/static/chunks/bb923814e1f4d2e9.js","/_next/static/chunks/dd3ee9f5b7716ca6.js","/_next/static/chunks/4204a0ae82542ca8.js","/_next/static/chunks/1270dead247da766.js","/_next/static/chunks/6e1aa89a642f40be.js","/_next/static/chunks/2d7aa753e81324a8.js","/_next/static/chunks/8ffd0b71567920d8.js","/_next/static/chunks/67ea6dfd21f16c5f.js","/_next/static/chunks/24eb4379f68013fd.js","/_next/static/chunks/b8cd775ffdf935bd.js","/_next/static/chunks/983103cd9eb7b428.js","/_next/static/chunks/263f2cdcb9aa2341.js","/_next/static/chunks/294b9525248c8e5f.js"],"Grid"] 1d:I[617291,["/_next/static/chunks/bb923814e1f4d2e9.js","/_next/static/chunks/dd3ee9f5b7716ca6.js","/_next/static/chunks/4204a0ae82542ca8.js","/_next/static/chunks/1270dead247da766.js","/_next/static/chunks/6e1aa89a642f40be.js","/_next/static/chunks/2d7aa753e81324a8.js","/_next/static/chunks/8ffd0b71567920d8.js","/_next/static/chunks/67ea6dfd21f16c5f.js","/_next/static/chunks/24eb4379f68013fd.js","/_next/static/chunks/b8cd775ffdf935bd.js","/_next/static/chunks/983103cd9eb7b428.js","/_next/static/chunks/263f2cdcb9aa2341.js","/_next/static/chunks/294b9525248c8e5f.js"],"GridCol"] 1e:I[734616,["/_next/static/chunks/bb923814e1f4d2e9.js","/_next/static/chunks/dd3ee9f5b7716ca6.js","/_next/static/chunks/4204a0ae82542ca8.js","/_next/static/chunks/1270dead247da766.js","/_next/static/chunks/6e1aa89a642f40be.js","/_next/static/chunks/2d7aa753e81324a8.js","/_next/static/chunks/8ffd0b71567920d8.js","/_next/static/chunks/67ea6dfd21f16c5f.js","/_next/static/chunks/24eb4379f68013fd.js","/_next/static/chunks/b8cd775ffdf935bd.js","/_next/static/chunks/983103cd9eb7b428.js","/_next/static/chunks/263f2cdcb9aa2341.js","/_next/static/chunks/294b9525248c8e5f.js"],"Avatar"] 1f:I[432972,["/_next/static/chunks/bb923814e1f4d2e9.js","/_next/static/chunks/dd3ee9f5b7716ca6.js","/_next/static/chunks/4204a0ae82542ca8.js","/_next/static/chunks/1270dead247da766.js","/_next/static/chunks/6e1aa89a642f40be.js","/_next/static/chunks/2d7aa753e81324a8.js","/_next/static/chunks/8ffd0b71567920d8.js","/_next/static/chunks/67ea6dfd21f16c5f.js","/_next/static/chunks/24eb4379f68013fd.js","/_next/static/chunks/b8cd775ffdf935bd.js","/_next/static/chunks/983103cd9eb7b428.js","/_next/static/chunks/263f2cdcb9aa2341.js","/_next/static/chunks/294b9525248c8e5f.js"],"default"] 20:T4d3,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.

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