3tk3 | pdb_00003tk3
Cytochrome P450 2B4 mutant L437A in complex with 4-(4-chlorophenyl)imidazole
Structural highlights
FunctionCP2B4_RABIT Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. In the epoxidation of arachidonic acid it has a unique preference for the 5,6-olefin. Publication Abstract from PubMedResidues located outside of 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 eleven sites were constructed, expressed in E. 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 approximately 20-times higher than wild type 2B4, and the K(s) of L437A for 4-CPI was approximately 50-times higher than wild type, leading to significantly altered inhibitor selectivity. Enzyme function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 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 yield 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. 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.,Wilderman PR, Gay SC, Jang HH, Zhang Q, Stout CD, Halpert JR FEBS J. 2011 Nov 3. doi: 10.1111/j.1742-4658.2011.08411.x. PMID:22051155[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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