The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0-Å Resolution

Abstract

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.P450 2C9 is one of three human microsomal cytochrome P450s (CYPs) 1 in subfamily 2C that contribute extensively to the hepatic metabolism of therapeutic drugs. The P450 2C9 locus is polymorphic leading to a diminished capacity to clear specific drugs in genetically affected individuals. For P450 2C9 substrates, such as warfarin or phenytoin, that have low therapeutic margins of safety, diminished metabolic capacity because of genetic polymorphisms or drug-drug interactions can lead to toxicity at normal therapeutic doses (1). P450 2C9 has also been implicated in the synthesis of arachidonic acid epoxides in extrahepatic tissues where they regulate blood pressure (2). Like other P450 subfamilies, the 2C enzymes share roughly 70% or greater amino acid identity. However, the 2C genes have duplicated and diverged rapidly as mammalian species evolved, leading to different numbers of enzymes in various species and highly divergent substrate selectivities. This diversity reflects high rates of non-synonymous substitutions that often alter residues that line the active site cavity and determine substrate selectivity.Human P450s 2C9 and 2C19 are closely related with roughly 91% amino acid identity. Although they exhibit distinct substrate selectivities, residues predicted to line the active site cavity, based on the published structures of other mammalian P450s (3-6), do not differ between the two enzymes. This suggests that conformation changes are likely to underlie differences in the substrate selectivities of P450s 2C9 and 2C19 and that the structure(s) of one or both will differ from those published previously. This is supported by studies of chimeric enzymes generated from P450s 2C9 and 2C19 that have generally identified amino acid residues that are predicted to reside outside the substrate binding cavity as determinants of their distinct catalytic properties (7-9). P450 2C9 exhibits a selectivity for the oxidation of relatively small, lipophilic anions such as the non-steroidal anti-inflammator...

Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain

“…Although a larger activation would probably be required to affect cholesterol turnover significantly in vivo, the current results serve as a proof of concept that activation of CYP46A1 is possible, in principle. The mechanism for this activation could be similar to that proposed for the stimulation of the CYP2C9-mediated 4Ј-hydroxylation of flurbiprofen by dapsone (32)(33)(34). The stimulation is suggested to occur via simultaneous binding of dapsone and flurbiprofen to the active site of CYP2C9 with dapsone binding limiting the motion of flurbiprofen and affecting the hydration of the active site.…”

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

“…The alternative conformation of the 2C9 B-C loop positions the conserved Arg-108 in the active site, where it forms an ionic bond with the carboxylate of flurbiprofen in the 2C9 structure (Protein Data Bank code 1R9O). This rearrangement alters both the polar properties and the shape of the active site cavity and underlies the role of 2C9 in the metabolic clearance of other small anionic drugs and the distinct contributions of the two enzymes to drug metabolism (79,82).…”

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

“…In the human genome, 57 genes have been found to code for CYPs [2]. The main CYPs implicated in drug metabolism, such as CYP3A4, CYP2C9 or CYP2D6, and those responsible for the biosynthesis of steroid hormones have been extensively studied, and several x-ray structures of human CYPs have been recently published [3][4][5][6][7][8][9][10]. Much less is known about more recently discovered human CYPs such as CYP2J2 [2,11,12].…”