Research Mission and Profile
Molecular Signal Processing
Bioorganic Chemistry
Biochemistry of Plant Interactions
Cell and Metabolic Biology
Independent Junior Research Groups
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Good Scientific Practice
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Publications
Cytochrome P450s are versatile catalysts for biosynthesis applications. In the P450 catalytic cycle, two electrons are required to reduce the heme iron and activate the subsequent reductions through proposed electron transfer pathways (eTPs), which often represent the rate-limiting step in reactions. Herein, the P450 BM3 from Bacillus megaterium was engineered for improved catalytic performance by redesigning proposed eTPs. By introducing aromatic amino acids on eTPs of P450 BM3, the “best” variant P2H02 (A399Y/Q403F) showed 13.9-fold improved catalytic efficiency (kcat/KM = 913.5 L mol−1 s−1) compared with P450 BM3 WT (kcat/KM = 65.8 L mol−1 s−1). Molecular dynamics simulations and electron hopping pathways analysis revealed that aromatic amino acid substitutions bridging the cofactor flavin mononucleotide and heme iron could increase electron transfer rates and improve catalytic performance. Moreover, the introduction of tyrosines showed positive effects on catalytic efficiency by potentially protecting P450 from oxidative damage. In essence, engineering of eTPs by aromatic amino acid substitutions represents a powerful approach to design catalytically efficient P450s (such as CYP116B3) and could be expanded to other oxidoreductases relying on long-range electron transfer pathways.
Publications
The combinatorial complexity of the protein sequence space presents a significant challenge for recombination experiments targeting beneficial positions. To overcome these difficulties, a machine learning (ML) approach was employed, which was trained on a limited literature dataset and combined with iterative generation and experimental data implementation. The PyPEF method was utilized to identify existing variants and predict recombinant variants targeting the substrate channel of P450 CYP116B3. Through molecular dynamics simulations, eight multiple-substituted improved variants were successfully validated. Specifically, the RMSF of variant A86T/T91H/M108S/A109M/T111P was decreased from 3.06 Å (wild type) to 1.07 Å. Additionally, the average RMSF of the variant A86T/T91P/M108V/A109M/T111P decreased to 1.41 Å, compared to the wild type’s 1.53 Å. Of particular significance was the prediction that the variant A86T/T91H/M108G/A109M/T111P exhibited an activity approximately 15 times higher than that of the wild type. Furthermore, during the selection of the regression model, PLS and MLP regressions were compared. The effect of data size and data relevance on the two regression approaches has been summarized. The aforementioned conclusions provide evidence for the feasibility of the strategy that combines ML with experimental approaches. This integrated strategy proves effective in exploring potential variations within the protein sequence space. Furthermore, this method facilitates a deeper understanding of the substrate channel in P450 CYP116B3.
