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Active Site Crowding of Cytochrome P450 3A4 as a Strategy To Alter Its Selectivity
Author(s) -
Schiavini Paolo,
Cheong Kin J.,
Moitessier Nicolas,
Auclair Karine
Publication year - 2017
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201600546
Subject(s) - steric effects , chemistry , active site , selectivity , hydroxylation , cytochrome p450 , stereochemistry , stereoselectivity , substrate (aquarium) , protein engineering , combinatorial chemistry , rational design , steroid , enzyme , biochemistry , catalysis , biology , hormone , ecology , genetics
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.