ArticleThe FEBS journal2012
Investigation by site-directed mutagenesis of the role of cytochrome P450 2B4 non-active-site residues in protein-ligand interactions based on crystal structures of the ligand-bound enzyme.
Article in The FEBS journal, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 31 citations in OpenAlex.
- Recombinant Technologies Facilitate Drug Metabolism, Pharmacokinetics, and General Biomedical Research.Drug metabolism and disposition: the biological fate of chemicals · 2023Review
- Tyrosine Nitration Contributes to Nitric Oxide-Stimulated Degradation of CYP2B6.Molecular pharmacology · 2020Article
- Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon.TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik · 2020Article
- Advances in the Understanding of Protein-Protein Interactions in Drug Metabolizing Enzymes through the Use of Biophysical Techniques.Frontiers in pharmacology · 2017Review
- Article
- Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism.Molecular pharmacology · 2016Article
- Functional importance of a peripheral pocket in mammalian cytochrome P450 2B enzymes.Archives of biochemistry and biophysics · 2015Article
- Substrate tunnels in enzymes: structure-function relationships and computational methodology.Proteins · 2015Review
- A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factories.Computational and structural biotechnology journal · 2014Review
- Human cytochrome P450 17A1 conformational selection: modulation by ligand and cytochrome b5.The Journal of biological chemistry · 2014Article
- Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida.Toxicology and applied pharmacology · 2014Article
- A structural snapshot of CYP2B4 in complex with paroxetine provides insights into ligand binding and clusters of conformational states.The Journal of pharmacology and experimental therapeutics · 2013Article
- Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
- Article
- Conformational adaptation of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 revealed upon binding multiple amlodipine molecules.Biochemistry · 2012Article
- Bioinformatics and variability in drug response: a protein structural perspective.Journal of the Royal Society, Interface · 2012Review
- A novel type of allosteric regulation: functional cooperativity in monomeric proteins.Archives of biochemistry and biophysics · 2012Review
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
Abstract
Residues located outside the active site of cytochromes P450 2B have exhibited importance in ligand binding, structural stability and drug metabolism. However, contributions of non-active-site residues to the plasticity of these enzymes are not known. Thus, a systematic investigation was undertaken of unique residue-residue interactions found in crystal structures of P450 2B4 in complex with 4-(4-chlorophenyl)imidazole (4-CPI), a closed conformation, or in complex with bifonazole, an expanded conformation. Nineteen mutants distributed over 11 sites were constructed, expressed in Escherichia coli and purified. Most mutants showed significantly decreased expression, especially in the case of interactions found in the 4-CPI structure. Six mutants (H172A, H172F, H172Q, L437A, E474D and E474Q) were chosen for detailed functional analysis. Among these, the K(s) of H172F for bifonazole was ∼ 20 times higher than for wild-type 2B4, and the K(s) of L437A for 4-CPI was ∼ 50 times higher than for wild-type, leading to significantly altered inhibitor selectivity. Enzyme function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin, 7-methoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin (7-BR). H172F was inactive with all three substrates, and L437A did not turn over 7-BR. Furthermore, H172A, H172Q, E474D and E474Q showed large changes in k(cat)/K(M) for each of the three substrates, in some cases up to 50-fold. Concurrent molecular dynamics simulations yielded distances between some of the residues in these putative interaction pairs that are not consistent with contact. The results indicate that small changes in the protein scaffold lead to large differences in solution behavior and enzyme function.
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