8GM2

Crystal structure of T252E-CYP199A4 in complex with 4-methoxybenzoic acid soaked with 2 mM hydrogen peroxide

Summary for 8GM2

• Entry DOI: 10.2210/pdb8gm2/pdb
• Descriptor: Cytochrome P450, 4-METHOXYBENZOIC ACID, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• Functional Keywords: cytochrome, bacterial p450, oxidoreductase
• Biological source: Rhodopseudomonas palustris HaA2
• Total number of polymer chains: 1
• Total formula weight: 45370.04

Authors

Lee, J.H.Z.,Bruning, J.B.,Bell, S.G. (deposition date: 2023-03-24, release date: 2023-12-27, Last modification date: 2024-02-21)

Primary citation

Lee, J.H.Z.,Bruning, J.B.,Bell, S.G.
An In Crystallo Reaction with an Engineered Cytochrome P450 Peroxygenase.
Chemistry, 30:e202303335-e202303335, 2024

PubMed Abstract: The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H O ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H O enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H O for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H O will enable new structural biology methods and techniques to be applied to study their mechanism of action.

PubMed: 37971151
DOI: 10.1002/chem.202303335

Experimental method

X-RAY DIFFRACTION (2.33 Å)