5DYZ

Crystal structure of Asp251Gly/Gln307His mutant of cytochrome P450 BM3 in complex with N-palmitoylglycine

Summary for 5DYZ

• Entry DOI: 10.2210/pdb5dyz/pdb
• Descriptor: Bifunctional P-450/NADPH-P450 reductase, PROTOPORPHYRIN IX CONTAINING FE, N-PALMITOYLGLYCINE, ... (4 entities in total)
• Functional Keywords: cytochrome p450, random mutagenesis, drug metabolism, substrate, oxidoreductase
• Biological source: Bacillus megaterium
• Cellular location: Cytoplasm : P14779
• Total number of polymer chains: 2
• Total formula weight: 108728.61

Authors

Di Nardo, G.,Dell'Angelo, V.,Gilardi, G. (deposition date: 2015-09-25, release date: 2016-01-20, Last modification date: 2024-01-10)

Primary citation

Di Nardo, G.,Dell'Angelo, V.,Catucci, G.,Sadeghi, S.J.,Gilardi, G.
Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen.
Arch.Biochem.Biophys., 602:106-115, 2016

PubMed Abstract: This paper reports the structure of the double mutant Asp251Gly/Gln307His (named A2) generated by random mutagenesis, able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. The 3D structure of the substrate-free mutant shows a conformation similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on I-helix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules. The impact of these structural features on thermal stability, reduction potential and electron transfer is investigated. The data demonstrate that a single mutation far from the active site triggers an increase in protein flexibility in a key region, shifting the conformational equilibrium toward the closed form that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.

PubMed: 26718083
DOI: 10.1016/j.abb.2015.12.005

Experimental method

X-RAY DIFFRACTION (1.967 Å)