Computational protocol: Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design

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Protocol publication

[…] The FRESCO method was employed to obtain a thermostabilized variant of HMFO. Computational modeling was performed using the 4UDQ X-ray structure of HMFO (1.6 Å resolution). To avoid mutations that could interfere with the active site, only residues that were > 5 Å away from FAD were mutated []. The computational selection was started with calculating the predicted change of free folding energy (ΔΔGFold) with FoldX ( and Rosetta-ddg ( [, , ]. For Rosetta, the so-called row-3 protocol (described by Kellogg et al. in row 3 of their table 1) was invoked using the following options: -ddg::weight_file soft_rep_design -ddg::iterations 50 -ddg::local_opt_only true -ddg::min_cst false -ddg::mean true -ddg::min false -ddg::sc_min_only false -ddg::ramp_repulsive false -ddg::opt_radius 8.0 []. For FoldX, the used options were --command=BuildModel --numberOfRuns=5. The single point mutations with a predicted ΔΔGFold < − 5 kJ mol−1 were subsequently submitted to MD simulations under Yasara as previously described [, ]. The averaged structures from the MD trajectories were visually inspected comparing the variant simulations with the wild-type HMFO while examining backbone and sidechain flexibility, hydrogen bonds, and hydrophobic exposure. This last in silico step is to further reduce the number of potentially thermostable variants to experimentally screen. All FRESCO specific scripts and code are available at […]

Pipeline specifications

Software tools FoldX, YASARA
Applications Drug design, Protein structure analysis
Chemicals Carbohydrates, Fructose