title: Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells.
abstract: Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich-Heine University, 40225 Düsseldorf, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Roellecke
authors.affiliation: Institute of Biochemistry, Heinrich-Heine University, 40225 Düsseldorf, Germany.
authors.forename: Vera D
authors.initials: VD
authors.lastname: Jäger
authors.affiliation: Institute of Biochemistry, Heinrich-Heine University, 40225 Düsseldorf, Germany.
authors.forename: Veselin H
authors.initials: VH
authors.lastname: Gyurov
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA 98195, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Kowalski
authors.affiliation: Institute of Biochemistry, Heinrich-Heine University, 40225 Düsseldorf, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Mielke
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA 98195, USA.
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich-Heine University, 40225 Düsseldorf, Germany.|Department of Pediatrics III, University Children's Hospital Essen, University of Duisburg-Essen, 45122 Essen, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich-Heine University, 40225 Düsseldorf, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Wiek
authors.affiliation: Institute of Biochemistry, Heinrich-Heine University, 40225 Düsseldorf, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Girhard
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056945:Hep G2 Cells; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1
keywords: 4-ipomeanol; CYP4B1; cytochrome P450 monooxygenase; perilla ketone; suicide gene