Structure of Human Microsomal Cytochrome P450 2C8

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.

[1]  James R. Halpert,et al.  An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[2]  E. Szczesna-Skorupa,et al.  Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells* , 2003, Journal of Biological Chemistry.

[3]  C David Stout,et al.  Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 A resolution: evidence for an induced fit model of substrate binding. , 2003, Biochemistry.

[4]  C David Stout,et al.  Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A resolution: evidence for multiple substrate binding modes. , 2003, Biochemistry.

[5]  Tetsuo Satoh,et al.  Tissue distribution of mRNA expression of human cytochrome P450 isoforms assessed by high-sensitivity real-time reverse transcription PCR. , 2003, Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan.

[6]  Dong-Sun Lee,et al.  Substrate Recognition and Molecular Mechanism of Fatty Acid Hydroxylation by Cytochrome P450 from Bacillus subtilis , 2003, The Journal of Biological Chemistry.

[7]  J. Imig,et al.  Kidney CYP450 enzymes: biological actions beyond drug metabolism. , 2003, Current drug metabolism.

[8]  Colin N. Dewey,et al.  Initial sequencing and comparative analysis of the mouse genome. , 2002 .

[9]  P. Neuvonen,et al.  Gemfibrozil inhibits CYP2C8-mediated cerivastatin metabolism in human liver microsomes. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[10]  R. Subramanian,et al.  Effects of fibrates on metabolism of statins in human hepatocytes. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[11]  B. Monsarrat,et al.  Regioselective metabolism of taxoids by human CYP3A4 and 2C8: structure-activity relationship. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[12]  Tommy B Andersson,et al.  Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate. , 2002, The Journal of pharmacology and experimental therapeutics.

[13]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[14]  Eric F. Johnson,et al.  Purification and crystallization of N-terminally truncated forms of microsomal cytochrome P450 2C5. , 2002, Methods in Enzymology.

[15]  J. Feely,et al.  Pharmacokinetic-Pharmacodynamic Drug Interactions with HMG-CoA Reductase Inhibitors , 2002, Clinical pharmacokinetics.

[16]  M. Lanotte,et al.  Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites. , 2000, Molecular pharmacology.

[17]  H. Yamazaki,et al.  A significant role of human cytochrome P450 2C8 in amiodarone N-deethylation: an approach to predict the contribution with relative activity factor. , 2000, Drug metabolism and disposition: the biological fate of chemicals.

[18]  David J. Schuller,et al.  Crystal Structure of a Thermophilic Cytochrome P450 from the Archaeon Sulfolobus solfataricus * , 2000, The Journal of Biological Chemistry.

[19]  Eric F. Johnson,et al.  Engineering Microsomal Cytochrome P450 2C5 to Be a Soluble, Monomeric Enzyme , 2000, The Journal of Biological Chemistry.

[20]  D E McRee,et al.  Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. , 2000, Molecular cell.

[21]  S. Clarke,et al.  Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone. , 2001, British journal of clinical pharmacology.

[22]  H. Yamazaki,et al.  Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[23]  K. Korzekwa,et al.  Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil. , 1999, British journal of clinical pharmacology.

[24]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[25]  J. Goldstein,et al.  Gene structure of CYP2C8 and extrahepatic distribution of the human CYP2Cs , 1999, Journal of biochemical and molecular toxicology.

[26]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[27]  T. Richardson,et al.  Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus. , 1997, Archives of biochemistry and biophysics.

[28]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[29]  T A Jones,et al.  Electron-density map interpretation. , 1997, Methods in enzymology.

[30]  T. Richardson,et al.  A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli. , 1995, Archives of biochemistry and biophysics.

[31]  R. DuBois,et al.  Molecular cloning, expression and characterization of an endogenous human cytochrome P450 arachidonic acid epoxygenase isoform. , 1995, Archives of biochemistry and biophysics.

[32]  A. Archakov,et al.  Dynamic interactions of rabbit liver cytochromes P450IA2 and P450IIB4 with cytochrome b5 and NADPH-cytochrome P450 reductase in proteoliposomes. , 1995, Biochemistry.

[33]  K. Korzekwa,et al.  Selective biotransformation of taxol to 6 alpha-hydroxytaxol by human cytochrome P450 2C8. , 1994, Cancer research.

[34]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[35]  S. Kawato,et al.  Rotation and membrane topology of genetically expressed methylcholanthrene-inducible cytochrome P-450IA1 lacking the N-terminal hydrophobic segment in yeast microsomes. , 1994, The Journal of biological chemistry.

[36]  G J Kleywegt,et al.  Detection, delineation, measurement and display of cavities in macromolecular structures. , 1994, Acta crystallographica. Section D, Biological crystallography.

[37]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[38]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[39]  H. Gelboin,et al.  Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies. , 1991, The Journal of biological chemistry.

[40]  Y. Fujii‐Kuriyama,et al.  Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes , 1978, The Journal of cell biology.

[41]  C. Jefcoate,et al.  Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. , 1978, Methods in enzymology.

[42]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. , 1964, The Journal of biological chemistry.