An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution

Abstract

The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-Å crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the ␣-helical and ␤-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetryrelated molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress. T he cytochromes P450 (P450s) are a superfamily of hemecontaining monooxygenases. They are responsible for the metabolism of an unusually wide range of endogenous and exogenous substrates, including synthesis of steroid hormones, bile acids, and cholesterol, and the degradation of steroids, fatty acids, drugs, toxins, and procarcinogens (1). P450s from families 1, 2, and 3 have evolved to convert lipophilic xenobiotics to more polar metabolites readily conjugated by phase II enzymes, and thus targeted for elimination. The stereo-and regiospecificity of metabolite formation by individual xenobiotic metabolizing P450s suggest very specific substrate-enzyme interactions, whereas the range of substrates metabolized suggests an induced fit type of substrate recognition. Understanding the basis for this specific, yet versatile, metabolism by mammalian xenobiotic metabolizing P450s has been limited by the dearth of structural information for these membrane proteins.In 2000, the first mammalian P450 structure was published (2, 3), that of P450 2C5, which was engineered to delete the single N-terminal transmembrane domain and to mutate a peripheral membrane-binding site. Recent structures of 2C5 bound with the substrates, diclofenac (4) and 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ) (5), indicate that flexible regions of the protein adapt for substrate binding, and that ligands may bind in multiple orientations. The 2C5 structures generally reveal the enzyme closed around these substrates wi...

A comparison of substrate dynamics in human CYP2E1 and CYP2A6

“…Supersomes™ (human CYP2E1 and human CYP2A6 with P450 reductase and cytochrome b5) and insect cell control microsomes were from BD Gentest. p-Xylene, p-xylene-α, α, α, α ′, α ′, α ′ -d 6 , methyl 3-methylbenzoate, methyl 4-methylbenzoate, lithium aluminum deuteride (LAD), methanesulfonyl chloride, N,Ndiisopropylethylamine (EDIPA), pentane, BSTFA, and NADPH, were purchased from SigmaAldrich. Methanol was purchased from Fisher.…”

“…Incubation conditions and analysis were the same as for the intramolecular experiments except that substrate consisted of a 1:1 mixture of unlabeled (d 0 ) and labeled (d 6 ; both methyl groups labeled) p-xylene which was added to a final total xylene concentration of 1 mM.…”

“…However, if association/dissociation is slow relative to the oxidative event then no isotope effect should be observed ((k H /k D ) obs = 1). Using labeled (d 6 ) and unlabeled (d 0 ) p-xylene as the substrate it is apparent that the rapid interconvertible model (rapid reorientation within the active site) is applicable for p-xylene interactions with both CYP2E1 and CYP2A6 as no isotope effect was observed for this substrate mixture.…”

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

“…To circumvent this problem, we replaced the amino terminus with a hydrophilic segment (MAKKTSS), which has been successfully used for protein crystallization of several members of P450 families 2 and 3 [15,18,19]. Additionally, a simplified purification procedure was developed to replace the previous scheme, which included ion exchange and hydrophobic columns.…”

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

“…Inherent in most of the models is the assumption that a loose fit of a single substrate molecule requires the binding of a second ligand for efficient binding and/or catalysis [9,10,12], although recent evidence for effector-induced conformational rearrangements has emerged from our laboratory and others [13][14][15]. Strong support for an important conformational transition in P450 function is provided by several recent x-ray crystal structures of mammalian P450 enzymes including CYP3A4, which have revealed considerable flexibility [11,[16][17][18].…”