An altered heme environment in an engineered cytochrome P450 enzyme enables the switch from monooxygenase to peroxygenase activity

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dc.contributor.authorPodgorski, M.N.
dc.contributor.authorHarbort, J.S.
dc.contributor.authorLee, J.H.Z.
dc.contributor.authorNguyen, G.T.H.
dc.contributor.authorBruning, J.B.
dc.contributor.authorDonald, W.A.
dc.contributor.authorBernhardt, P.V.
dc.contributor.authorHarmer, J.R.
dc.contributor.authorBell, S.G.
dc.date.issued2022
dc.description.abstractCytochrome P450 heme-thiolate monooxygenases are exceptionally versatile enzymes which insert an oxygen atom into the unreactive C–H bonds of organic molecules. They source O2 from the atmosphere and usually derive electrons from nicotinamide cofactors via electron transfer proteins. The requirement for an expensive nicotinamide adenine dinucleotide (phosphate) cofactor and the redox protein partners can be bypassed by driving the catalysis using hydrogen peroxide (H₂O₂). We demonstrate that the mutation of a highly conserved threonine residue, involved in dioxygen activation, to a glutamate shuts down monooxygenase activity in a P450 enzyme and converts it into a peroxygenase. The reason for this switch in the threonine to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas palustris HaA2 was linked to the lack of a spin state change upon the addition of the substrate. The crystal structure of the substrate-bound form of this mutant highlighted a modified oxygen-binding groove in the I-helix and the retention of the iron-bound aqua ligand. This ligand interacts with the glutamate residue, which favors its retention. Electron paramagnetic resonance confirmed that the ferric heme aqua ligand of the mutant substrate-bound complex had altered characteristics compared to a standard ferric heme aqua complex. Significant improvements in peroxygenase activity were demonstrated for the oxidative demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and veratric acid to vanillic acid (up to 6-fold). The detailed characterization of this engineered heme peroxygenase will facilitate the development of new methods for driving the biocatalytic generation of oxygenated organic molecules via selective C–H bond activation using heme enzymes.
dc.description.statementofresponsibilityMatthew N. Podgorski, Joshua S. Harbort, Joel H.Z. Lee, Giang T.H. Nguyen, John B. Bruning, William A. Donald, Paul V. Bernhardt, Jeffrey R. Harmer, and Stephen G. Bell
dc.identifier.citationACS Catalysis, 2022; 12(3):1614-1625
dc.identifier.doi10.1021/acscatal.1c05877
dc.identifier.issn2155-5435
dc.identifier.issn2155-5435
dc.identifier.orcidPodgorski, M.N. [0000-0003-3238-8735]
dc.identifier.orcidLee, J.H.Z. [0000-0001-6288-9034]
dc.identifier.orcidBruning, J.B. [0000-0002-6919-1824]
dc.identifier.orcidBell, S.G. [0000-0002-7457-9727]
dc.identifier.urihttps://hdl.handle.net/2440/134378
dc.language.isoen
dc.publisherAmerican Chemical Society (ACS)
dc.relation.granthttp://purl.org/au-research/grants/arc/DP140103229
dc.relation.granthttp://purl.org/au-research/grants/arc/DP190103158
dc.relation.granthttp://purl.org/au-research/grants/arc/DP160102681
dc.relation.granthttp://purl.org/au-research/grants/arc/FT140100355
dc.relation.granthttp://purl.org/au-research/grants/arc/FT120100421
dc.relation.granthttp://purl.org/au-research/grants/arc/FT200100798
dc.rights© 2022 American Chemical Society
dc.source.urihttps://doi.org/10.1021/acscatal.1c05877
dc.subjectBiocatalysis; heme enzymes; peroxygenases; protein engineering; dioxygen activation; X-ray crystallography; metalloenzymes
dc.titleAn altered heme environment in an engineered cytochrome P450 enzyme enables the switch from monooxygenase to peroxygenase activity
dc.typeJournal article
pubs.publication-statusPublished

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