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Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol
Constanze Wiek1, Eva M Schmidt2, Katharina Roellecke1
1*Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University, Moorenstrasse 5, D-40225, Düsseldorf, Germany.
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.
Insights
Human CYP4B1 enzymes activate toxins like 4-ipomeanol (4-IPO), unlike the rabbit version. Re-engineering human CYP4B1 enhances its stability and 4-IPO activation, creating a potential suicide gene system for cancer therapy.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Mammalian CYP4B1 enzymes are crucial cytochrome P450 mono-oxygenases involved in activating exogenous pro-toxins, such as 4-ipomeanol (4-IPO).
- Differences in CYP4B1 orthologs, like human vs. rabbit, impact their catalytic functions and substrate specificities.
- Understanding these differences is key to developing targeted therapies and bioactivation strategies.
Purpose of the Study:
- To investigate the functional differences in 4-IPO bioactivation between human and rabbit CYP4B1 enzymes.
- To identify key amino acid residues and regions responsible for the catalytic activity and stability of CYP4B1 enzymes.
- To engineer a novel human CYP4B1-based suicide gene system for potential use in adoptive cellular therapies.
Main Methods:
- Comparative analysis of native human CYP4B1 and orthologous rabbit CYP4B1 enzymes using 4-IPO as a substrate.
- Site-directed mutagenesis to probe the role of specific amino acid residues (e.g., S427P) in enzyme stability and activity.
- Systematic substitution of human CYP4B1 regions with corresponding rabbit CYP4B1 sequences.
- Development and characterization of a re-engineered human CYP4B1 mutant (P+12) for enhanced 4-IPO bioactivation.
Main Results:
- Native human CYP4B1 with serine at position 427 does not bioactivate 4-IPO or cause cytotoxicity.
- A proline residue at position 427 in human CYP4B1 rescues 4-IPO bioactivation and is crucial for protein stability.
- Identifying 18 key amino acids in rabbit CYP4B1 responsible for high 4-IPO metabolizing activity.
- A re-engineered human CYP4B1 (P+12) mutant, incorporating 12 specific amino acid changes, exhibits stability and activity comparable to wild-type rabbit CYP4B1.
- These mutations cluster in the B-C loop through F-helix regions, highlighting their importance for P450 stability.
Conclusions:
- The proline residue at position 427 is critical for human CYP4B1 stability and 4-IPO bioactivation.
- Specific amino acid substitutions, particularly in the B-C loop to F-helix regions, can significantly enhance human CYP4B1 activity and stability.
- A minimally re-engineered human CYP4B1 enzyme (P+12) demonstrates efficient 4-IPO activation, paving the way for a novel human suicide gene system for cancer therapy.
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