title: High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.
abstract: The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, 31077 Toulouse, France ; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400 Toulouse, France ; CNRS, UMR 5504, 135 Avenue de Rangueil, 31400 Toulouse, France.
authors.forename: Philippe
authors.identifier: 0000-0003-0557-839X
authors.initials: P
authors.lastname: Urban
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, 31077 Toulouse, France ; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400 Toulouse, France ; CNRS, UMR 5504, 135 Avenue de Rangueil, 31400 Toulouse, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Truan
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, 31077 Toulouse, France ; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400 Toulouse, France ; CNRS, UMR 5504, 135 Avenue de Rangueil, 31400 Toulouse, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Pompon
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008861:Microsomes; D015735:Mifepristone; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000081223:Polycyclic Sesquiterpenes; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D012717:Sesquiterpenes; D013256:Steroids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000081223:Polycyclic Sesquiterpenes; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; D013256:Steroids; D015735:Mifepristone; D003577:Cytochrome P-450 Enzyme System; C050302:nootkatone