8YAZ

XFEL crystal structure of the oxidized form of F87A/F393H P450BM3 with N-enanthyl-L-prolyl-L-phenylalanine in complex with styrene

Summary for 8YAZ

• Entry DOI: 10.2210/pdb8yaz/pdb
• Descriptor: Bifunctional cytochrome P450/NADPH--P450 reductase, PROTOPORPHYRIN IX CONTAINING FE, (2S)-2-[[(2S)-1-heptylpyrrolidin-2-yl]carbonylamino]-3-phenyl-propanoic acid, ... (6 entities in total)
• Functional Keywords: cytochrome p450, oxidoreductase
• Biological source: Priestia megaterium NBRC 15308 = ATCC 14581
• Total number of polymer chains: 2
• Total formula weight: 106607.21

Authors

Nagao, S.,Kuwano, W.,Tosha, T.,Yamashita, K.,Stanfield, J.K.,Kasai, C.,Ariyasu, S.,Shoji, O.,Sugimoto, H.,Kubo, M. (deposition date: 2024-02-11, release date: 2025-02-12, Last modification date: 2025-04-02)

Primary citation

Nagao, S.,Kuwano, W.,Tosha, T.,Yamashita, K.,Stanfield, J.K.,Kasai, C.,Ariyasu, S.,Hirata, K.,Ueno, G.,Murakami, H.,Ago, H.,Yamamoto, M.,Shoji, O.,Sugimoto, H.,Kubo, M.
XFEL crystallography reveals catalytic cycle dynamics during non-native substrate oxidation by cytochrome P450BM3.
Commun Chem, 8:63-63, 2025

PubMed Abstract: Cytochrome P450s are haem-containing enzymes, catalysing the regio- and stereospecific oxidation of non-activated hydrocarbons. Among these, the bacterial P450BM3 is a promising biocatalyst due to its high enzymatic activity. Given the significant conformational flexibility of this enzyme, understanding protein-substrate interactions and associated structural dynamics are crucial for designing P450BM3-based biocatalysts. Herein, employing an X-ray free electron laser in combination with freeze-trap crystallography and spectroscopy techniques, we captured the intact structures of engineered P450BM3s in the initial stages of catalysis during styrene epoxidation, in the presence of a decoy molecule. We found that the iron reduction significantly altered the active-site orientation of styrene, driven by structural changes in surrounding helices and hydrogen-bonding networks. Oxygen binding to iron further stabilised its productive orientation, providing a molecular basis for the experimentally observed enzyme kinetics and enantioselectivities. This study reveals the substrate dynamics of a P450 enzyme, showcasing how changes in haem chemistry affect enzyme structure and substrate orientation.

PubMed: 40075209
DOI: 10.1038/s42004-025-01440-2

Experimental method

X-RAY DIFFRACTION (1.85 Å)