Abstract
Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the β3-β4 strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates.
Highlights
The cytochrome P450 (CYP) 3A subfamily accounts for ~30% of the total CYP content in adult liver and plays a major role in drug metabolism [1]
The wild type (WT) protein does not produce crystals, even in the presence of small heme-ligating molecules, such as imidazole, phenyl-imidazole, or dimethyl sulfoxide, known to stabilize and promote CYP crystallization. This is in contrast to ∆3-22 CYP3A4, which willingly crystallizes in the ligand-free and ligand-bound forms
CYP3A4 crystal packing was analyzed to identify divergent surface residues in Cytochrome P450 3A7 (CYP3A7) that could potentially interfere with crystal formation
Summary
The cytochrome P450 (CYP) 3A subfamily accounts for ~30% of the total CYP content in adult liver and plays a major role in drug metabolism [1]. This subfamily includes four enzymes: CYP3A4, CYP3A5, CYP3A7 and CYP3A43. Since the wild type (WT) CYP3A7 resists crystallization, site-directed mutagenesis of surface residues was conducted to find a variant that promotes crystal growth One such variant, R69G/C77G/K244E/K421A/K422A/K424A (CYP3A7mut), was produced, and its 2.15 Å dithiothreitol-bound structure was solved, providing the first insights into the molecular architecture and enabling comparative analysis of CYP3A enzymes. Based on our data and the previously reported functional results, we propose that decreased conformational plasticity and higher rigidity of the active site are the main reason for the diminished ligand binding and catalytic ability of CYP3A7
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