Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

• Published in: Archives of biochemistry and biophysics 2016-07, Vol.602, p.106-115
• Main Authors: Di Nardo, Giovanna, Dell'Angelo, Valentina, Catucci, Gianluca, Sadeghi, Sheila J., Gilardi, Gianfranco
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - ultrastructure; Binding Sites; Biocatalysis; Computer Simulation; Conformational equilibrium; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - ultrastructure; Cytochrome P450; Diclofenac - chemistry; Enzyme Activation; Ibuprofen - chemistry; Molecular Docking Simulation - methods; Molecular Sequence Data; Mutation - genetics; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - genetics; NADPH-Ferrihemoprotein Reductase - ultrastructure; Protein Binding; Protein Conformation; Protein engineering; Structure-Activity Relationship; Tolbutamide - chemistry
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2015.12.005

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. [Display omitted] •The crystal structure of Asp251Gly/Gln307His mutant of cytochrome P450 BM3 was solved.•The mutations allow a different conformational equilibrium to establish.•The mutations have structural long range effect on the protein active site.•The mutations alter the reduction potential and first electron transfer rate.•The engineered mutations alter both protein structure and function.