Title: Structure of Human Microsomal Cytochrome P450 2C8
Abstract: A 2.7-Å molecular structure of human microsomal cytochrome P450 2C8 (CYP2C8) was determined by x-ray crystallography. The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the β1 sheet and helix F′ that have not previously been implicated in substrate recognition by mammalian P450s. CYP2C8 crystallized as a symmetric dimer formed by the interaction of helices F, F′, G′, and G. Two molecules of palmitic acid are bound in the dimer interface. The dimer is observed in solution, and mass spectrometry confirmed the association of palmitic acid with the enzyme. This novel finding identifies a peripheral binding site in P450s that may contribute to drug-drug interactions in P450 metabolism. A 2.7-Å molecular structure of human microsomal cytochrome P450 2C8 (CYP2C8) was determined by x-ray crystallography. The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the β1 sheet and helix F′ that have not previously been implicated in substrate recognition by mammalian P450s. CYP2C8 crystallized as a symmetric dimer formed by the interaction of helices F, F′, G′, and G. Two molecules of palmitic acid are bound in the dimer interface. The dimer is observed in solution, and mass spectrometry confirmed the association of palmitic acid with the enzyme. This novel finding identifies a peripheral binding site in P450s that may contribute to drug-drug interactions in P450 metabolism. Xenobiotic metabolizing cytochrome P450 monooxygenases provide crucial protection from the harmful effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. In general, these microsomal enzymes determine the bioavailability of hydrophobic compounds by controlling the rate of conversion to more soluble, inactive products that are readily excreted. Different P450s can show overlapping substrate specificities, and individual enzymes can interact with numerous structurally diverse substrates. This broad catalytic activity usually serves a positive defensive role; however, in some cases, it can lead to adverse drug-drug interactions. P450s often possess enzyme-specific catalytic repertoires and can display exquisite catalytic selectivity for regio- and stereospecific reactions. This is particularly evident for the mammalian family 2C P450s, which exhibit extensive, independent evolution and functional divergence in mammals, leading to multiple enzymes in each species while retaining a high degree of amino acid identity (>70%). Thus, structural comparisons within this highly related but functionally diverse P450 subfamily are likely to be particularly revealing of mechanisms leading to the catalytic diversity of P450 enzymes. The structural features of drug-metabolizing P450s that contribute to their capacity to oxidize structurally diverse substrates while maintaining site-specific oxidation is an active area of research because of the obvious benefits that this information could provide regarding xenobiotic risk assessment, the prediction and avoidance of negative drug-drug interactions, as well as the rational design of improved therapeutic drugs and specific inhibitors. Herein, we report the first structure of a catalytically active, human microsomal cytochrome P450 2C8 (CYP2C8) 1The abbreviations used are: P450 or CYP, generic terms for cytochrome P450 monooxygenases. Individual P450 enzymes are denoted by a number/letter/number combination derived from a uniform nomenclature based on sequence identity. obtained without the use of multiple internal mutations to the catalytic domain. Analysis of this structure suggests several general mechanisms for altering the topography and function of the P450 active site. These include residue substitutions that affect the volume available for substrates, regional flexibility to enhance substrate interactions, and peripheral binding sites that can modulate active site characteristics and enable dimerization to further restrict localized adaptive changes that can influence substrate binding and oxidation. Human CYP2C8 plays a central role in the metabolism of a number of therapeutic drugs. This enzyme is expressed at relatively high levels in the liver (1Klose T.S. Blaisdell J.A. Goldstein J.A. J. Biochem. Mol. Toxicol. 1999; 13: 289-295Crossref PubMed Scopus (167) Google Scholar, 2Nishimura M. Yaguti H. Yoshitsugu H. Naito S. Satoh T. Yakugaku Zasshi. 2003; 123: 369-375Crossref PubMed Scopus (267) Google Scholar), which is often the principal site for drug clearance. CYP2C8 has been shown to contribute extensively to the clearance of the anticancer drug taxol (3Rahman A. Korzekwa K.R. Grogan J. Gonzalez F.J. Harris J.W. Cancer Res. 1994; 54: 5543-5546PubMed Google Scholar, 4Cresteil T. Monsarrat B. Dubois J. Sonnier M. Alvinerie P. Gueritte F. Drug Metab. Dispos. 2002; 30: 438-445Crossref PubMed Scopus (92) Google Scholar), the antimalarial drug amodiaquine (5Li X.Q. Bjorkman A. Andersson T.B. Ridderstrom M. Masimirembwa C.M. J. Pharmacol. Exp. Ther. 2002; 300: 399-407Crossref PubMed Scopus (239) Google Scholar), the antidiabetic drugs troglitazone (6Yamazaki H. Shibata A. Suzuki M. Nakajima M. Shimada N. Guengerich F.P. Yokoi T. Drug Metab. Dispos. 1999; 27: 1260-1266PubMed Google Scholar) and rosiglitazone (7Baldwin S.J. Clarke S.E. Chenery R.J. Br. J. Clin. Pharmacol. 1999; 48: 424-432Crossref PubMed Scopus (218) Google Scholar), the anti-arrhythmic drug amiodarone (8Ohyama K. Nakajima M. Nakamura S. Shimada N. Yamazaki H. Yokoi T. Drug Metab. Dispos. 2000; 28: 1303-1310PubMed Google Scholar), and the calcium channel blocker verapamil (9Tracy T.S. Korzekwa K.R. Gonzalez F.J. Wainer I.W. Br. J. Clin. Pharmacol. 1999; 47: 545-552Crossref PubMed Scopus (155) Google Scholar). CYP2C8 is the primary enzyme that metabolizes a cholesterol lowering drug cerivastatin, and inhibition of its metabolism by a CYP2C8 inhibitor, gemfibrozil, a lipid lowering drug, causes a toxic drug-drug interaction that provokes rhabdomyolysis (10Prueksaritanont T. Tang C. Qiu Y. Mu L. Subramanian R. Lin J.H. Drug Metab. Dispos. 2002; 30: 1280-1287Crossref PubMed Scopus (297) Google Scholar, 11Wang J.S. Neuvonen M. Wen X. Backman J.T. Neuvonen P.J. Drug Metab. Dispos. 2002; 30: 1352-1356Crossref PubMed Scopus (164) Google Scholar, 12Williams D. Feely J. Clin. Pharmacokinet. 2002; 41: 343-370Crossref PubMed Scopus (383) Google Scholar). CYP2C8 is also involved in the metabolism of natural substrates like unsaturated fatty acids and retinoic acid (13Marill J. Cresteil T. Lanotte M. Chabot G.G. Mol. Pharmacol. 2000; 58: 1341-1348Crossref PubMed Scopus (175) Google Scholar). In human liver and kidney, CYP2C8 is the predominant P450 responsible for the oxidation of arachidonic acid to generate biologically active epoxyecosatrienoic acids that are involved in the regulation of blood pressure (14Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (156) Google Scholar, 15Zhao X. Imig J.D. Curr. Drug Metab. 2003; 4: 73-84Crossref PubMed Scopus (84) Google Scholar). The ability to determine structures for the human drug-metabolizing P450s has been hindered by the inherent difficulties associated with crystallizing membrane enzymes. Recently, our laboratory engineered rabbit microsomal CYP2C5 for expression in Escherichia coli as a conditionally soluble membrane protein that retained catalytic activity (16Cosme J. Johnson E.F. J. Biol. Chem. 2000; 275: 2545-2553Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Crystallization of the protein led to the first structure of a microsomal P450 (17Williams P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-132Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). Similar modifications to the N terminus allowed us to express the native sequence of the CYP2C8 catalytic domain for structural studies. The experimentally determined structure diverges significantly from the structure of CYP2C5 in ways that are critical for function. The major structural differences contribute to a much larger substrate binding site cavity in CYP2C8 that is compatible with its capacity to oxidize relatively large substrates such as taxol. Surprisingly, CYP2C8 crystallized as a symmetric dimer formed by the interaction of the helix F to G regions of the protein. Two molecules of palmitic acid are bound in the dimer interface. This novel finding suggests the existence of a peripheral binding site that can affect the structural dynamics of the active site and may underlie the cooperative effects of drug-drug interactions in P450 metabolism. Construction and Purification of CYP2C8dH—The CYP2C8 cDNA used for modifications was kindly provided by Robert Tukey (University of California, San Diego). The CYP2C8 cDNA was modified by replacing the sequence encoding the first 27 amino acids with one encoding a short hydrophilic, positively charged N terminus (MAKKTSSKG) identical to that employed for the expression and crystallization of CYP2C5 (16Cosme J. Johnson E.F. J. Biol. Chem. 2000; 275: 2545-2553Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Von Wachenfeldt C. Richardson T.H. Cosme J. Johnson E.F. Arch. Biochem. Biophys. 1997; 339: 107-114Crossref PubMed Scopus (105) Google Scholar). This modification of the N terminus of CYP2C8dH converts the integral membrane protein to one that binds peripherally to membranes and that can be separated from the membrane by elevating the ionic strength of the medium. In addition, a four-residue histidine tag was added to the C terminus to facilitate purification. The modified cDNA was cloned into the pCWori plasmid for expression in E. coli. The resulting enzyme is designated CYP2C8dH. The modified enzyme was isolated in the presence of the detergent CYMAL5 (Anatrace, Maumee) as described for CYP2C5dH (19Wester M.R. Stout C.D. Johnson E.F. Methods Enzymol. 2002; 357: 73-79Crossref PubMed Scopus (31) Google Scholar) with some modifications. Briefly, CYP2C8dH was purified from E. coli lysates by metal ion affinity column chromatography (nickel-nitrilotriacetic acid, Qiagen Inc., Valencia, CA). Following elution of the protein in a buffer containing 40 mm histidine, the protein was subjected to CM-Sepharose ion exchange chromatography (CL6-B, Amersham Biosciences, Piscataway, NJ) to deplete the detergent. The purified protein was concentrated using a centrifugal device (Ultrafree-15 50K, Millipore, Billerica). CYP2B4dH and CYP2C9dH constructs were expressed and purified in a similar way as CYP2C8dH. The expression vector for 2B4dH (20Scott E.E. He Y.A. Wester M.R. White M.A. Chin C.C. Halpert J.R. Johnson E.F. Stout C.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13196-13201Crossref PubMed Scopus (347) Google Scholar) was kindly provided by Emily Scott and Jim Halpert (University of Texas Medical Branch). Characterization of CYP2C8dH—The purified protein exhibited a specific P450 content of 18 nmol/mg of protein, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein indicated a molecular mass of 53,988 Da, which agrees with the 53,979 Da predicted from the amino acid composition of CYP2C8dH. The concentration of P450 was determined as described previously (21Omura T. Sato R. J. Biol. Chem. 1964; 239: 2379-2385Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined using bicinchoninic acid reagent (Pierce Biotechnology, Rockford, IL). Based on an analysis of type I difference spectra (22Jefcoate C.R. Methods Enzymol. 1978; 52: 258-279Crossref PubMed Scopus (394) Google Scholar), purified CYP2C8dH binds arachidonic acid (Sigma-Aldrich, St. Louis, MO) with an apparent KD of 1.8 μm with a maximum 27% conversion of the enzyme to the high spin form. This binding constant is 6-fold lower than values measured previously for the partially purified wild type enzyme (14Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (156) Google Scholar). Taxol also produced a partial conversion of the enzyme to the high spin form, but the limited solubility of this substrate precluded an estimate of the binding affinity. The rate of 6α-hydroxylation of taxol (10 μm) was determined to be 0.5 nmol/min/nmol of P450, when CYP2C8dH (10 pmol) was reconstituted with 0.28 unit (1 μmol of cytochrome c reduced/min/mg) of P450 reductase in 50 mm HEPES, pH 7.5, in the presence of 30 μg of dilaurylphosphatidylcholine in a total volume of 1 ml at 37 °C for 15 min. Product formation was analyzed by high-performance liquid chromatography as described (23Richardson T.H. Jung F. Griffin K.J. Wester M. Raucy J.L. Kemper B. Bornheim L.M. Hassett C. Omiecinski C.J. Johnson E.F. Arch. Biochem. Biophys. 1995; 323: 87-96Crossref PubMed Scopus (142) Google Scholar) using 6α-hydroxytaxol (Gentest) as a quantitative standard. The substrate was not soluble at higher concentrations precluding a determination of Vmax. Crystallization and Data Collection—The protein was crystallized by the vapor diffusion method using 2.5-μl sitting drops containing 1.25 μl of 392 μm P450, 1.12 mm CYMAL-6 detergent (Anatrace, Maumee), 40 mm potassium phosphate, pH 7.4, 400 mm NaCl, 0.8 mm EDTA, 0.16 mm dithiothreitol, and 16% glycerol and 1.25 μl of 100 mm HEPES, pH 7.5, 15% ethanol, and 10% PEG4000. The drops were equilibrated with 100 mm HEPES, pH 7.5, 15% ethanol, and 10% PEG4000 at 24 °C. For data collection, the crystal was soaked for 2 min in a cryoprotectant composed of 70 mm HEPES, pH 7.5, 10.5% ethanol, 7% PEG4000, and 30% ethylene glycol, and then flash-frozen in liquid N2 prior to transfer into the cryo-stream for data collection at Stanford Synchrotron Radiation Laboratory beam line 7-1. The data were collected at 100 K for a single crystal of dimensions 0.15 × 0.15 × 0.4 mm using a MAR345 image plate (Mar Research) with 2° oscillations (50 frames, 300-s exposure). The crystal did not decay noticeably during data collection. The data were processed with CCP4 programs MOSFLM and SCALA (24Leslie A.G.W. A Data Collection Strategy Option in MOSFLM, CCP4 Newsletter.(www.ccp4.ac.uk/newsletter/mosflm.html)Date: 1996Google Scholar, 25CCP4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). CNS (version 1.1) (26Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) was used for initial phasing by molecular replacement and subsequent refinement protocols. Xfit/Xtalview (27McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar) and O (28Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (505) Google Scholar) were employed for display electron density maps and model building. Structure Determination—The structure of CYP2C5/3LVdH (PDB ID code 1N6B) was used as the template for model building via molecular replacement. Divergent residues were truncated to Cβ in the search model. Molecular replacement was carried out with CNS (version 1.1) (26Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Standard refinement protocols were employed, and the refinement proceeded normally through several cycles of interpretation, editing, and adjustment of the model into σA-weighted 2 Fo - Fc and Fo - Fc (where Fo = observed structure factor and Fc = calculated structure factor) electron density maps using either Xfit/Xtalview (27McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar) or O (28Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (505) Google Scholar). Following rebuilding, subsequent models were refined, using CNS, by simulated annealing (using non-crystallographic symmetry restraints in the first rounds), and individual atomic, isotropic B-factor refinement. In addition to small individual adjustments of backbone and side chain, the peptide backbone for residues 207-217 (F/G loop) and 271-277 (polar surface loop) were entirely rebuilt, and one palmitic acid was added per protein molecule in the asymmetric unit and adjusted into the observed density. Extraction and Characterization of Fatty Acids Associated with CYP2C8dH—Tetradeuterated palmitic acid (5 nmol) (Medical Isotopes) was added to 10 nmol of different preparations of purified P450s for use as an internal standard. After an incubation with 0.5 nmol of proteinase K, 1 volume of 10 mm NaOH was added to facilitate the release of bound fatty acid. After a second addition of 1 volume of 100 mm NaOH, the pH was decreased with an excess of HCl. Samples were extracted with 2.5 volumes of organic phase (ether/hexane, 50/50), dried under nitrogen, reconstituted in methanol, and analyzed by electrospray ionization mass spectrometry (PerkinElmer Life Sciences API 100 Sciex single quadrupole). Structure Determination—The structure of CYP2C8dH was determined from data collected for a single crystal diffracting to 2.7 Å. The enzyme crystallized in the C2 space group with two molecules per asymmetric unit. The structure was solved initially by molecular replacement using the structure of CYP2C5/3LVdH (PDB ID code 1N6B) (29Wester M.R. Johnson E.F. Marques-Soares C. Dansette P.M. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 6370-6379Crossref PubMed Scopus (207) Google Scholar). The initial maps were highly interpretable, and the overall structure is very similar to that of CYP2C5/3LVdH. However, additional refinement and rebuilding were necessary to construct divergent regions. This included portions of the polypeptide backbone that form the active site cavity. The final models for both molecules in the asymmetric unit exhibited connective electron density for residues 28-490, where the numbering corresponds to that of the native enzyme, with an R-factor of 24.7% (Rfree 28.4%) (Table I). Backbone geometry was analyzed in PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and only one residue (Gln278 in molecule B) was in the disallowed region of the Ramachandran plot. The two molecules of CYP2C8dH in the asymmetric unit are highly similar with an root mean square deviation of 0.52 Å (for 461 Cα atoms) and average B values of 60.2 Å2 and 65.8 Å2, respectively, for the A and B molecules.Table IData collection and refinement statisticsP450 construct2C8dHNo. of crystals1ComplexPalmitateSpace groupC2Unit cell (a, b, c, β) (E)105.99, 137.41, 97.32 (112.51°)Data collection and refinementWavelength (Å)1.08Resolution range (Å)50.0−2.70Total observations79,013Unique reflections > 0.0 σF34,573Completeness %aValues for the highest resolution shell are in parentheses.97.5 (97.9)〈I/σI〉aValues for the highest resolution shell are in parentheses.10.9 (1.3)Rsymm (I)aValues for the highest resolution shell are in parentheses.0.055 (0.512)R-factor0.247Rfree (5% of data)0.284Root mean square deviation bonds (Å)0.0091Root mean square deviation angles (deg)bRamachandran plot: 84.7% of residues in most favored regions; 13.9% in allowed regions; 1.2% in generously allowed regions; 0.1% in disfavored regions.1.60Model residuesNo. of atomsAvg. B-factor (Å2)ProteincResidues 28-490 in molecules A and B of the CYP2C8dH dimer.7,39063.2Heme8644.1Palmitate3660.5Water3751.7a Values for the highest resolution shell are in parentheses.b Ramachandran plot: 84.7% of residues in most favored regions; 13.9% in allowed regions; 1.2% in generously allowed regions; 0.1% in disfavored regions.c Residues 28-490 in molecules A and B of the CYP2C8dH dimer. Open table in a new tab Crystallization of CYP2C8dH as a Dimer Stabilized by Fatty Acids Bound in the Interface—The two molecules in the asymmetric unit form a symmetric dimer with the regions between helices F to G of molecules A and B interacting extensively (Fig. 1A). CYP2C8dH exhibits the typical P450 fold illustrated in Fig. 1B. Following the completion and building of the protein model, additional electron density was present within the dimer interface (Fig. 1C) that suggested the presence of two fatty acid molecules. A palmitic acid molecule, a C16 saturated fatty acid, was built into a long, almost continuous, tubular density with a branched terminus that resides near the N-terminal end of helix G of the dimerization partner. The average B values are slightly lower for the two palmitic acids (60.5 Å2) than the average values exhibited by the protein molecules (63.2 Å2) (Table I). The terminal portion of the aliphatic chain of the palmitic acid resides in a tubular, hydrophobic cavity formed by helices F′, G′, and G of each protein molecule (Fig. 1A). The carboxylate group of the fatty acid interacts with the backbone amides in the first turn of helix G of the second molecule in the dimer (Fig. 1C). Thus, the binding of the ionized fatty acid is likely to be stabilized by the helix dipole as well as by direct hydrogen bonding interactions with the backbone amides forming the first turn of helix G. A portion of the aliphatic chains of the two fatty acids are exposed to each other and are separated by roughly 5 Å, which adds additional hydrophobic surface area to the interaction at the dimer interface. Size exclusion chromatography indicates that the isolated protein is predominantly a dimer in solution with evidence for a small amount of monomer (Fig. 2). The dimer tends to dissociate at low P450 concentrations. Ether/hexane extracts of the enzyme following proteolytic digestion and neutralization were characterized by electrospray ionization mass spectrometry (Fig. 3). The chromatograms revealed a molecular ion with a mass that is consistent with the presence of palmitic acid, m/z 255. A second, prominent molecular ion, at m/z 283, could represent the C18 fatty acid, stearic acid. These ions were not observed in extracts from other P450s that were expressed and purified in a similar manner. It is likely that the fatty acid molecules were acquired in the E. coli host used for expression of the protein, and palmitic and stearic acid are the predominant saturated fatty acids in E. coli. The density observed for the fatty acid (Fig. 1C) is consistent with the 16 carbon aliphatic chain of palmitic acid. However, partial occupation of the site by stearic acid cannot be excluded.Fig. 3Mass spectral evidence for bound fatty acid. Negative ion electrospray mass spectra of ether/hexane extracts from 10 nmol of CYP2C8dH and CYP2C9dH are shown. Tetradeuterated-palmitic acid (5 nmol, peak 259 Da) was added to the proteins prior to extraction to provide an internal standard. Two peaks that correspond to m/z values for palmitate (255 Da) and stearate (283 Da) are present in the CYP2C8dH extract. These are not observed in extracts from CYP2C9dH, CYP2B4dH, or buffer-only samples (not shown). The molar ratio of bound fatty acid from three different purified CYP2C8dH preparations ranges from 35 to 77%, with a constant molar ratio of palmitic/stearic acid of 1.9 ± 0.1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Comparison of the 2C8 and 2C5 Active Sites—Examination of the structure of CYP2C8dH indicates that the active site cavity is roughly twice as large as that of CYP2C5/3LVdH (Fig. 4). This is consistent with the capacity of CYP2C8 to oxidize larger substrates such as taxol. The increased volume of the active site results not only from changes in the conformation of portions of the protein that include helices F′ and B′, which delimit the distal portion of the active site cavity, but also from differences in the volume of side chains that occupy the cavity. The smaller volume of the CYP2C5 active site largely reflects the presence of two juxtaposed phenylalanine residues, Phe114 and Phe473 (Fig. 4). In CYP2C5, Leu213 of helix F′ is packed behind these two residues, and together they partition that section of the binding site cavity in CYP2C5. In CYP2C8, the corresponding residues are smaller, Ser114 and Ile476, and the distance between side chains increases from 5.6 Å (closest atom center to closest atom center) in CYP2C5 to 13.5 Å in CYP2C8. The size of the active site cavity is expanded through the opening between residues 114 and 476, because helix F′ of CYP2C8 is positioned up and away from these two residues, which results in a significantly larger active site cavity. This reflects a 9-Å difference in the position of the Trp212 Cα atom at the corner between helices F and F′ (Fig. 4C). Additional factors that contribute to the larger active site volume of CYP2C8 are the altered position of the B′ helix, which differs by roughly 2.4 Å from CYP2C5, and the side chains of the residues on the B′-helix that project into the substrate binding cavity are smaller in CYP2C8 than the corresponding residues in CYP2C5. The structure of CYP2C8 reported here illustrates several structural features of the xenobiotic metabolizing P450s that reflect the rapid genetic and functional diversification of these enzymes between mammalian species. Although the mammalian CYP2C enzymes generally exhibit greater than 70% amino acid identity and a higher degree of amino acid similarity, the genes encoding these enzymes exhibit extensive, independent duplication and diversification in mammalian species. As a consequence, the number of CYP2C enzymes varies among species, and each enzyme exhibits a distinct spectrum of substrate and inhibitor selectivity. This appears to reflect a high rate of non-synonymous nucleotide substitutions that alter amino acids that form the substrate binding site and contribute to the capacity to oxidize and detoxify a wide range of xenobiotics (31Waterston R.H. Lindblad-Toh K. Birney E. Rogers J. Abril J.F. Agarwal P. Agarwala R. Ainscough R. Alexandersson M. An P. Antonarakis S.E. Attwood J. Baertsch R. Bailey J. Barlow K. Beck S. Berry E. Birren B. Bloom T. Bork P. Botcherby M. Bray N. Brent M.R. Brown D.G. Brown S.D. Bult C. Burton J. Butler J. Campbell R.D. Carninci P. Cawley S. Chiaromonte F. Chinwalla A.T. Church D.M. Clamp M. Clee C. Collins F.S. Cook L.L. Copley R.R. Coulson A. Couronne O. Cuff J. Curwen V. Cutts T. Daly M. David R. Davies J. Delehaunty K.D. Deri J. Dermitzakis E.T. Dewey C. Dickens N.J. Diekhans M. Dodge S. Dubchak I. Dunn D.M. Eddy S.R. Elnitski L. Emes R.D. Eswara P. Eyras E. Felsenfeld A. Fewell G.A. Flicek P. Foley K. Frankel W.N. Fulton L.A. Fulton R.S. Furey T.S. Gage D. 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