title: Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen.
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.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Via Accademia Albertina 13, Torino, Italy; CrisDi, Interdepartmental Center for Crystallography, University of Torino, Via Pietro Giuria 7, Torino, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Di Nardo
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Via Accademia Albertina 13, Torino, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Dell'Angelo
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Via Accademia Albertina 13, Torino, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Catucci
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Via Accademia Albertina 13, Torino, Italy.
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Via Accademia Albertina 13, Torino, Italy; CrisDi, Interdepartmental Center for Crystallography, University of Torino, Via Pietro Giuria 7, Torino, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D001665:Binding Sites; D003198:Computer Simulation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D004008:Diclofenac / Q000737:chemistry*; D004789:Enzyme Activation; D007052:Ibuprofen / Q000737:chemistry*; D062105:Molecular Docking Simulation / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014044:Tolbutamide / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D007052:Ibuprofen
keywords: Biocatalysis; Conformational equilibrium; Cytochrome P450; Protein engineering