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- Title
Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells.
- Authors
Roellecke, Katharina; Gyurov, Veselin H.; Kowalski, John P.; Mielke, Stephanie; Rettie, Allan E.; Hanenberg, Helmut; Wiek, Constanze; Girhard, Marco
- 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 wildtype 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.
- Subjects
ESCHERICHIA coli; CYTOCHROME P-450; LIGANDS (Biochemistry); MONOOXYGENASES; ENZYME metabolism; T cells; THERAPEUTICS
- Publication
PEDS: Protein Engineering, Design & Selection, 2017, Vol 30, Issue 3, p205
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzw075