We investigated the 16 -hydroxylation of steroid molecules and regioselective binding mode in homology-modeled cytochrome P450-2C11 to correlate the biological study with the computational molecular modeling. It revealed that there was a positive relationship between the observed inhibitory potencies and the binding free energies. Docking of steroid molecules into this homology-modeled CYP2C11 indicated that 16 -hydroxylation is favored with steroidal molecules possessing the following components, (1) a bent A-B ring configuration (5 -reduced), (2) C-3 -hydroxyl group, (3) C-17 -acetyl group, and (4) methyl group at both the C-18 and C-19. These respective steroid components requirements were defined as the inhibitory contribution factor. Overall studies of the male rat CYP2C11 metabolism revealed that the above-mentioned steroid components requirements were essential to induce an effective inhibition of [3H]progesterone 16 -hydroxylation. As far as docking of homology-modeled CYP2C11 against investigated steroids is concerned, they are docked at the active site superimposed with flurbiprofen. It was also found that the distance between heme iron and C16 -H was between 4 to 6？？ and that the related angle was in the range of . 1. Introduction Cytochrome P450 (P450) constitutes a large superfamily of heme-containing enzymes capable of oxidizing a variety of substrates, both of endogenous (such as steroids) and exogenous (xenobiotics) origins [1–7]. Although a variety of P450s are able to metabolize a broad range of substrates, the enzymes often exhibit strict regio- and stereoselectivity towards pertinent compounds, such as various steroids [1]. One of the most active and versatile P450 is rat CYP2C11, a microsomal P450 isoform catalyzing more than 90% of steroid 16 -hydroxylations [8–10]. It is well-known that several 3-keto-4-ene steroids such as progesterone and testosterone are metabolized in a gender-specific and predominant manner by the adult rat liver microsomes. In the male, these steroids are primarily metabolized into two oxidized (16 -hydroxyl and 6 -hydroxyl) products mainly by the respective, male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they are primarily metabolized into the 5 -reduced products by female predominant 5 -reductase [11]. Most of P450 structures reveal that the heme group is buried deep within the protein matrix, indicating that residues outside of the active site may also be required to guide the substrate into the heme pocket by recognizing substrates at the protein surface and/or comprising part of a
References
[1]
Z. K. Ali, R. J. Kim, and F. M. Ysla, “CYP2C9 polymorphisms: considerations in NSAID therapy,” Current Opinion in Drug Discovery and Development, vol. 12, no. 1, pp. 108–114, 2009.
[2]
J. B. Hagan, R. L. Taylor, R. J. Singh, J. C. Seegmiller, B. C. Netzel, and N. L. Korpi-Steiner, Fluticasone Propionate and Its Metabolite in Urine of Human for Treatment Compliance Assessment, Mayo Foundation for Medical Education and Research, 2009.
[3]
Y. S. Kang, S. Y. Park, C. H. Yim et al., “The CYP3A4*18 genotype in the cytochrome P450 3A4 gene, a rapid metabolizer of sex steroids, is associated with low bone mineral density,” Clinical Pharmacology and Therapeutics, vol. 85, no. 3, pp. 312–318, 2009.
[4]
V. Luu-The, D. Duche, C. Ferraris, J.-R. Meunier, J. Leclaire, and F. Labrie, “Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin？ and full thickness model from Episkin？,” Journal of Steroid Biochemistry and Molecular Biology, vol. 116, no. 3–5, pp. 178–186, 2009.
[5]
A. Mahmood, A. Kachurin, W. Warren, and R. Higbee, Artificial Tissue Constructs Comprising Alveolar Cells Epithelial and Endothelial Layers and Methods for Using the Same as in Vitro Models of Mammalian Lung Tissue for Study of Lung Diseases, Vaxdesign, Minneapolis, Minn, USA, 2009.
[6]
L. M. Scott, P. Durant, S. Leone-Kabler et al., “Effects of prior oral contraceptive use and soy isoflavonoids on estrogen-metabolizing cytochrome P450 enzymes,” Journal of Steroid Biochemistry and Molecular Biology, vol. 112, no. 4-5, pp. 179–185, 2008.
[7]
Y.-Y. Zhang and L. Yang, “Interactions between human cytochrome P450 enzymes and steroids: physiological and pharmacological implications,” Expert Opinion on Drug Metabolism and Toxicology, vol. 5, no. 6, pp. 621–629, 2009.
[8]
G. D. Szklarz, R. L. Ornstein, and J. R. Halpert, “Application of 3-dimensional homology modeling of cytochrome P450 2B1 for interpretation of site-directed mutagenesis results,” Journal of Biomolecular Structure and Dynamics, vol. 12, no. 1, pp. 61–78, 1994.
[9]
G. A. Dannan, F. P. Guengerich, and D. J. Waxman, “Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of Δ4-steroid 5α-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450,” Journal of Biological Chemistry, vol. 261, no. 23, pp. 10728–10735, 1986.
[10]
S. Imaoka, S. Fujita, and Y. Funae, “Age-dependent expression of cytochrome P-450s in rat liver,” Biochimica et Biophysica Acta, vol. 1097, no. 3, pp. 187–192, 1991.
[11]
J. B. Schenkman, “Steroid metabolism by constitutive cytochromes P450,” Journal of Steroid Biochemistry and Molecular Biology, vol. 43, no. 8, pp. 1023–1030, 1992.
[12]
W. Li, H. Liu, E. E. Scott et al., “Possible pathway(s) of testosterone egress from the active site of cytochrome P450 2B1: a steered molecular dynamics simulation,” Drug Metabolism and Disposition, vol. 33, no. 7, pp. 910–919, 2005.
[13]
G. D. Szklarz and J. R. Halpert, “Use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships of mammalian cytochromes P450,” Life Sciences, vol. 61, no. 26, pp. 2507–2520, 1997.
[14]
S. Kumar, E. E. Scott, H. Liu, and J. R. Halpert, “A rational approach to re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5,” Journal of Biological Chemistry, vol. 278, no. 19, pp. 17178–17184, 2003.
[15]
M. R. Wester, E. F. Johnson, C. Marques-Soares, P. M. Dansette, D. Mansuy, and C. D. Stout, “Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 ？ resolution: evidence for multiple substrate binding modes,” Biochemistry, vol. 42, no. 21, pp. 6370–6379, 2003.
[16]
M. R. Wester, E. F. Johnson, C. Marques-Soares et al., “Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 ？ resolution: evidence for an induced fit model of substrate binding,” Biochemistry, vol. 42, no. 31, pp. 9335–9345, 2003.
[17]
P. A. Williams, J. Cosme, V. Sridhar, E. F. Johnson, and D. E. McRee, “Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity,” Molecular Cell, vol. 5, no. 1, pp. 121–131, 2000.
[18]
T. Schwede, J. Kopp, N. Guex, and M. C. Peitsch, “SWISS-MODEL: an automated protein homology-modeling server,” Nucleic Acids Research, vol. 31, no. 13, pp. 3381–3385, 2003.
[19]
N. Guex and M. C. Peitsch, “SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling,” Electrophoresis, vol. 18, no. 15, pp. 2714–2723, 1997.
[20]
P. A. Williams, J. Cosme, A. Ward, H. C. Angove, D. M. Vinkovi？, and H. Jhoti, “Crystal structure of human cytochrome P450 2C9 with bound warfarin,” Nature, vol. 424, no. 6947, pp. 464–468, 2003.
[21]
M. R. Wester, J. K. Yano, G. A. Schoch et al., “The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-？ resolution,” Journal of Biological Chemistry, vol. 279, no. 34, pp. 35630–35637, 2004.
[22]
G. A. Schoch, J. K. Yano, M. R. Wester, K. J. Griffin, C. D. Stout, and E. F. Johnson, “Structure of human microsomal cytochrome P450 2C8: evidence for a peripheral fatty acid binding site,” Journal of Biological Chemistry, vol. 279, no. 10, pp. 9497–9503, 2004.
[23]
E. E. Scott, Y. A. He, M. R. Wester et al., “An open conformation of mammalian cytochrome P450 2B4 at 1.6-？ resolution,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 23, pp. 13196–13201, 2003.
[24]
J. K. Yano, M.-H. Hsu, K. J. Griffin, C. D. Stout, and E. F. Johnson, “Structures of human microsomal cytochrome P450 2A6 complexed with coumarin and methoxsalen,” Nature Structural and Molecular Biology, vol. 12, no. 9, pp. 822–823, 2005.
[25]
J. K. Yano, M. R. Wester, G. A. Schoch, K. J. Griffin, C. D. Stout, and E. F. Johnson, “The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-？ resolution,” Journal of Biological Chemistry, vol. 279, no. 37, pp. 38091–38094, 2004.
[26]
P. A. Williams, J. Cosme, D. Matak Vinkovi？ et al., “Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone,” Science, vol. 305, no. 5684, pp. 683–686, 2004.
[27]
L. M. Podust, L. V. Yermalitskaya, G. I. Lepesheva, V. N. Podust, E. A. Dalmasso, and M. R. Waterman, “Estriol bound and ligand-free structures of sterol 14α-demethylase,” Structure, vol. 12, no. 11, pp. 1937–1945, 2004.
[28]
L. M. Podust, T. L. Poulos, and M. R. Waterman, “Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 6, pp. 3068–3073, 2001.
[29]
M. D. Paulsen and R. L. Ornstein, “Predicting the product specificity and coupling of cytochrome P450cam,” Journal of Computer-Aided Molecular Design, vol. 6, no. 5, pp. 449–460, 1992.
[30]
M. Yamada, K. Indo, T. Nishigami, K. Nakasho, and H. Miyaji, “Progesterone-binding site of adult male rat liver microsomes,” Journal of Biological Chemistry, vol. 265, no. 19, pp. 11035–11043, 1990.
[31]
M. Yamada, T. Nishigami, K. Nakasho, Y. Nishimoto, and H. Miyaji, “Relationship between sigma-like site and progesterone-binding site of adult male rat liver microsomes,” Hepatology, vol. 20, no. 5, pp. 1271–1280, 1994.
[32]
A. Yamada, M. Yamada, Y. Fujita, T. Nishigami, K. Nakasho, and K. Uematsu, “Self-augmentation effect of male-specific products on sexually differentiated progesterone metabolism in adult male rat liver microsomes,” Journal of Biological Chemistry, vol. 276, no. 7, pp. 4604–4610, 2001.
[33]
M. Yamada and K. Matsumoto, “Pathway from progesterone to 5α reduced C19 steroids not involving androstenedione and testosterone in immature rat testes in vitro,” Endocrinology, vol. 94, no. 3, pp. 777–784, 1974.
[34]
F. De Rienzo, F. Fanelli, M. C. Menziani, and P. G. De Benedetti, “Theoretical investigation of substrate specificity for cytochromes P450 IA2, P450 IID6 and P450 IIIA4,” Journal of Computer-Aided Molecular Design, vol. 14, no. 1, pp. 93–116, 2000.
[35]
G. M. Morris, D. S. Goodsell, R. S. Halliday et al., “Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function,” Journal of Computational Chemistry, vol. 19, no. 14, pp. 1639–1662, 1998.
