title: Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol.
abstract: Mammalian CYP4B1 enzymes are cytochrome P450 mono-oxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast with the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine residue at position 427 is unable to bioactivate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYP4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B-C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans.
authors.affiliation: *Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University, Moorenstrasse 5, D-40225, Düsseldorf, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Wiek
authors.affiliation: †Department of Pediatric Hematology/Oncology, Children's Hospital, Heinrich Heine University, Moorenstrasse 5, D-40225, Düsseldorf, Germany.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Schmidt
authors.affiliation: *Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University, Moorenstrasse 5, D-40225, Düsseldorf, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Roellecke
authors.affiliation: *Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University, Moorenstrasse 5, D-40225, Düsseldorf, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Freund
authors.affiliation: ‡Departments of Medicinal Chemistry and Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA 98195, U.S.A.
authors.forename: Mariko
authors.initials: M
authors.lastname: Nakano
authors.affiliation: ‡Departments of Medicinal Chemistry and Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA 98195, U.S.A.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Kelly
authors.affiliation: §Center for Bioinformatics and Biostatistics (CBiBs), Center of Biological and Medical Research (BMFZ), Heinrich Heine University, Moorenstrasse 5, D-40225, Düsseldorf, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kaisers
authors.affiliation: ║Department of Physiology and Membrane Biology, University of California, Davis, CA 95616, U.S.A.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Yarov-Yarovoy
authors.affiliation: ¶Division of Pediatric Hematology and Oncology, Department of Child and Adolescent Health, University Medical Center Göttingen, Robert-Koch-Strasse 40, DE-37075, Göttingen, Germany.
authors.forename: Christof M
authors.initials: CM
authors.lastname: Kramm
authors.affiliation: ‡Departments of Medicinal Chemistry and Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA 98195, U.S.A.
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
authors.affiliation: *Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University, Moorenstrasse 5, D-40225, Düsseldorf, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D041722:Genes, Transgenic, Suicide; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D011392:Proline / Q000378:metabolism*; D015202:Protein Engineering; D011817:Rabbits; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism* / Q000633:toxicity
chemical_list: D013729:Terpenes; D011392:Proline; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1; C001470:4-ipomeanol