UPO-Mimetic Engineering of CYP199A4 Enables Enhanced Peroxygenase Activity via the Hydrogen Peroxide Shunt Pathway
P. Velazquez Garcia (TU Delft - BT/Biocatalysis)
T. Hilberath (TU Delft - BT/Biocatalysis)
P.L. Hagedoorn (TU Delft - BT/Biocatalysis)
F. Hollmann (TU Delft - BT/Biocatalysis)
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Abstract
Engineering an acid–base dyad into the peroxygenase-enabled mutant CYP199A4T252E yielded four in silico-designed double mutants, of which CYP199A4F182R/T252E showed the best dyad-like geometry and was characterized further. It delivered 10-fold higher initial H2O2-driven O-demethylation activity than wild type and CYP199A4T252E, alongside reduced catalase activity and improved peroxide utilization. However, it was more prone to H2O2-induced heme bleaching and rapid inactivation under standard dosing; slow, controlled H2O2 feeding sustained catalysis for hours. Overall, adding a second basic residue boosts per-oxy-gen-ase-like activity but reduces oxidative robustness, underscoring the trade-off between efficiency and peroxide tolerance and guiding future engineering of robust P450 peroxygenases.