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Publication Abstract from PubMed

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.

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen.,Di Nardo G, Dell'Angelo V, Catucci G, Sadeghi SJ, Gilardi G Arch Biochem Biophys. 2015 Dec 21. pii: S0003-9861(15)30116-8. doi:, 10.1016/j.abb.2015.12.005. PMID:26718083^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.