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Applied Biochemistry and Biotechnology

, Volume 186, Issue 1, pp 132–144 | Cite as

Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants

  • Amelia Nathania Dong
  • Yan Pan
  • Uma Devi Palanisamy
  • Beow Chin Yiap
  • Nafees Ahemad
  • Chin Eng Ong
Article

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.

Keywords

Cytochrome P450 Genetic polymorphism Protein expression Site-directed mutagenesis Spectral assays 

Notes

Acknowledgements

We express our gratitude to the Monash University Malaysia (Monash Seed Grant under the Bioactive Compounds Research Strength), the Ministry of Science, Technology & Innovation (grant no. 02-02-10-SF0077), and the Ministry of Higher Education (grant no. FRGS/1/2014/SKK03/MUSM/02/1) for funding and supporting this project.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12010_2018_2728_Fig4_ESM.gif (3.9 mb)
Fig. 1S

Representative reduced CO difference spectra showing expression of the (a) CYP2D6 wild type (CYP2D6*1) together with its alleles, and (b) CYP2C19 wild type (CYP2C19*1) with its alleles in E. coli membranes. (GIF 4009 kb)

12010_2018_2728_MOESM1_ESM.tif (527 kb)
High resolution image (TIFF 527 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of PharmacyMonash University MalaysiaBandar SunwayMalaysia
  2. 2.Department of Biomedical ScienceUniversity of Nottingham Malaysia CampusSemenyihMalaysia
  3. 3.School of PharmacyInternational Medical UniversityBukit JalilMalaysia

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