Invited Talk 21st International Conference on Biological Inorganic Chemistry 2025

Engineering peroxygenase activity into cytochrome P450 enzymes for selective C-H bond hydroxylations (#86)

Stephen G Bell 1 , Matthew N Podgorski 1 , Joel H.Z. Lee 1 , Jinia Akter 1 , Alecia R Gee 1 2 , Tuhin Das 1 , Oghenesivwe Osiebe 1 , Eva F Hayball 1 , Keith E Shearwin 3 , Fiona Whelan 3
  1. Department of Chemistry, University Adelaide, Adelaide, SA, Australia
  2. University of Adelaide, University Of Adelaide, SA, Australia
  3. School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia

Cytochrome P450 heme containing metalloenzymes (CYPs) are monooxygenases which catalyse a diverse range of oxidation reactions through the activation of dioxygen. However, the high cost of the required nicotinamide cofactors and their need for additional electron transfer proteins limits their use as biocatalysts in larger-scale chemical applications. We have recently identified that a family of these CYP enzymes involved in the oxidation of lignin derived aromatics can function as peroxygenases. We have investigated whether other bacterial CYPs can be converted into efficient hydrogen peroxide using peroxygenases through protein engineering of the enzyme’s oxygen activation machinery to more closely resemble those of the natural peroxygenase.1,2 We have developed mutants which have significantly higher peroxygenase activity than a single mutant prototype and that function at significantly lower hydrogen peroxide concentrations.3 The X-ray crystal structures and other spectroscopic techniques (UV-Vis/EPR) revealed significant structural changes at the heme and the oxygen-binding groove providing a rationale for the modified activity. In crystallo reactions have been undertaken by soaking crystals with hydrogen peroxide before data collection and exhibit loss or gain of electron density corresponding to the expected metabolite.4 We have extended our mutagenesis strategy to CYP enzymes from a diverse range of bacteria, and generated new peroxygenase biocatalysts for the regio- and stereo-selective hydroxylation of steroids, norisoprenoids and drug molecules at relatively low hydrogen peroxide concentrations.5-7 When this method is applied to CYP enzymes from thermophilic bacteria these reactions can occur at elevated temperatures enabling enzymatic hydroxylation reactions on a variety of substrates under non-standard biological conditions.7

 

  1. Podgorski, M. N.; Harbort, J. S.; Lee, J. H. Z.; Nguyen, G. T. H.; Bruning, J. B.; Donald, W. A.; Bernhardt, P. V.; Harmer, J. R.; Bell, S. G. ACS Catal. 2022, 12 (3), 1614-1625.
  2. [a] Harlington, A. C.; Shearwin, K. E.; Bell, S. G.; Whelan, F. Chem. Commun. 2022, 58, 13321–13324. [b] Harlington, A. C.; Das. T. ; :Shearwin, K. E.; Bell, S. G.; Whelan, F. Nat. Commun. 2025, 16, 1815.
  3. [a] Podgorski, M. N.; Akter, J. ; Churchman, L.R.; Bruning, J. B.; De Voss, J.J.; Bell, S. G. ACS Catal.2024 14 (10), 7426-7443 [b] Podgorski, M. N.; Bell, S. G. ACS Catal. 2025 15 (6), 5191–5210
  4. Lee, J. H. Z.; Bruning, J. B.; Bell, S. G. Chem. Eur. J. 2024, 30, e202303335.
  5. Lee, J.; Podgorski, M.; Moir, M.; Gee, A. R.; Bell, S. G. Chem. Eur. J. 2022, 28, e202201366.
  6. Akter, J.; Hayball, E. F.; Bell, S. G. Catal. Sci. Technol. 2023, 13, 6355-6359.
  7. [a] Gee, A. R.; Stone, I. S. J.; Stockdale, T. P.; Pukala, T. L.; De Voss, J. J.; Bell, S. G. Chem. Commun. 2023, 59, 13486–13489. [b] Das. T ; Harlington, A. C.; Das. T.; Hayball E. F.; Bell, S. G. ChemBioChem, 2025, 26 (2) e202400737