title: Active Site Crowding of Cytochrome P450 3A4 as a Strategy To Alter Its Selectivity.
abstract: Substrate-promiscuous enzymes are a promising starting point for the development of versatile biocatalysts. In this study, human cytochrome P450 3A4, known for its ability to metabolise hundreds of drugs, was engineered to alter its regio- and stereoselectivity. Rational mutagenesis was used to introduce steric hindrance in a specific manner in the large active site of P450 3A4 and to favour oxidation at a more sterically accessible position on the substrate. Hydroxylation of a synthetic precursor of (R)-lisofylline, a compound under investigation for its anti-inflammatory properties, was chosen as a first proof-of-principle application of our protein engineering strategy. In a second example, increasing active site crowding led to an incremental shift in the selectivity of oxidation from an internal double bond to a terminal phenyl group in a derivative of theobromine. The same correlation between crowding and selectivity was found in a final case focused on the hydroxylation of the steroid sex hormone progesterone.
authors.affiliation: Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8, Canada.
authors.forename: Paolo
authors.initials: P
authors.lastname: Schiavini
authors.affiliation: Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8, Canada.
authors.forename: Kin J
authors.initials: KJ
authors.lastname: Cheong
authors.affiliation: Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8, Canada.
authors.forename: Nicolas
authors.identifier: 0000-0001-6933-2079
authors.initials: N
authors.lastname: Moitessier
authors.affiliation: Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8, Canada.
authors.forename: Karine
authors.identifier: 0000-0003-0933-7956
authors.initials: K
authors.lastname: Auclair
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D010431:Pentoxifylline / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D011374:Progesterone; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C025189:lisofylline; D010431:Pentoxifylline
keywords: CYP 3A4; biocatalysis; crowding; cytochromes; mutagenesis