Metabolic Activity of Cytochrome P450 Expressed in Yeast

Ghanayem, B I.

Abstract

of Work: Significant variations in the metabolism of various drugs and environmental chemicals which are metabolized via cytochrome P450 (CYP) enzymes exist between humans. Many of these interindividual variations are attributed to polymorphisms in the CYP2C subfamily of enzymes. Current work focused on investigating the functional significance of specifically bioengineered CYP enzymes towards the metabolism of various substrates. Human recombinant CYP2C proteins for 2C8, 2C9, 2C18, and 2C19 were expressed in yeast or bacterial expression systems. An HPLC method for the quantitative determination of the hydroxylation of the nonsteroidal anti-inflammatory drug, diclofenac, by these enzymes was developed in our laboratory. Hydroxylation of diclofenac by 2C19 was approximately 100fold greater that that by 2C8, 2C18, or 2C19. The metabolic activities of various chimeric constructs of 2C9 and 2C19 were generated to determine regions within the 2C9 which confered diclofenac hydroxylation. These data indicated that SRS4 of CYP2C9 confers specificity for diclofenac hydroxylation.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Institute of Environmental Health Sciences (NIEHS)
• Type: Intramural Research (Z01)
• Project #: 1Z01ES048006-04
• Application #: 6162199
• Study Section: Special Emphasis Panel (LPC)
• Support Year: 4
• Fiscal Year: 1997

Institution

• Name: National Institute of Environmental Health Sciences
• Country: United States

Publications

Lee, Su-Jun; Bell, Douglas A; Coulter, Sherry J et al. (2005) Recombinant CYP3A4*17 is defective in metabolizing the hypertensive drug nifedipine, and the CYP3A4*17 allele may occur on the same chromosome as CYP3A5*3, representing a new putative defective CYP3A haplotype. J Pharmacol Exp Ther 313:302-9

Blaisdell, Joyce; Jorge-Nebert, Lucia F; Coulter, Sherry et al. (2004) Discovery of new potentially defective alleles of human CYP2C9. Pharmacogenetics 14:527-37

Lee, Su-Jun; Usmani, Khawja A; Chanas, Brian et al. (2003) Genetic findings and functional studies of human CYP3A5 single nucleotide polymorphisms in different ethnic groups. Pharmacogenetics 13:461-72

Blaisdell, Joyce; Mohrenweiser, Harvey; Jackson, Jonathan et al. (2002) Identification and functional characterization of new potentially defective alleles of human CYP2C19. Pharmacogenetics 12:703-11

Tsao, C C; Wester, M R; Ghanayem, B et al. (2001) Identification of human CYP2C19 residues that confer S-mephenytoin 4'-hydroxylation activity to CYP2C9. Biochemistry 40:1937-44

Ibeanu, G C; Blaisdell, J; Ferguson, R J et al. (1999) A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-mephenytoin. J Pharmacol Exp Ther 290:635-40