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- Title
Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants.
- Authors
Dong, Amelia Nathania; Pan, Yan; Palanisamy, Uma Devi; Yiap, Beow Chin; Ahemad, Nafees; Ong, Chin Eng
- Abstract
Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
- Subjects
SITE-specific mutagenesis; GENETIC polymorphisms; CYTOCHROME P-450 genetics; ENZYME kinetics; GENE expression; HUMAN genetic variation
- Publication
Applied Biochemistry & Biotechnology, 2018, Vol 186, Issue 1, p132
- ISSN
0273-2289
- Publication type
Article
- DOI
10.1007/s12010-018-2728-0