Biotransformation, Using Recombinant CYP450-Expressing Baker's Yeast Cells, Identifies a Novel CYP2D6.10A122V Variant Which Is a Superior Metabolizer of Codeine to Morphine Than the Wild-Type Enzyme

Williams, Ibidapo S.; Gatchie, Linda; Bharate, Sandip B.; Chaudhuri, Bhabatosh

Abstract

© Copyright 2018 American Chemical Society. CYP2D6, a cytochrome P450 (CYP) enzyme, metabolizes codeine to morphine. Within the human body, 0-15% of codeine undergoes O-demethylation by CYP2D6 to form morphine, a far stronger analgesic than codeine. Genetic polymorphisms in wild-type CYP2D6 (CYP2D6-wt) are known to cause poor-to-extensive metabolism of codeine and other CYP2D6 substrates. We have established a platform technology that allows stable expression of human CYP genes from chromosomal loci of baker's yeast cells. Four CYP2D6 alleles, (i) chemically synthesized CYP2D6.1, (ii) chemically synthesized CYP2D6-wt, (iii) chemically synthesized CYP2D6.10, and (iv) a novel CYP2D6.10 variant CYP2D6-C (i.e., CYP2D6.10A122V) isolated from a liver cDNA library, were cloned for chromosomal integration in yeast cells. When expressed in yeast, CYP2D6.10 enzyme shows weak activity compared with CYP2D6-wt and CYP2D6.1 which have moderate activity, as reported earlier. Surprisingly, however, the CYP2D6-C enzyme is far more active than CYP2D6.10. More surprisingly, although CYP2D6.10 is a known low metabolizer of codeine, yeast cells expressing CYP2D6-C transform >70% of codeine to morphine, which is more than twice that of cells expressing the extensive metabolizers, CYP2D6.1, and CYP2D6-wt. The latter two enzymes predominantly catalyze formation of codeine's N-demethylation product, norcodeine, with >55% yield. Molecular modeling studies explain the specificity of CYP2D6-C for O-demethylation, validating observed experimental results. The yeast-based CYP2D6 expression systems, described here, could find generic use in CYP2D6-mediated drug metabolism and also in high-yield chemical reactions that allow the formation of regio-specific dealkylation products.

• Journal Article Type: Article
• Acceptance Date: Aug 1, 2018
• Online Publication Date: Aug 9, 2018
• Publication Date: Aug 31, 2018
• Deposit Date: Aug 7, 2018
• Publicly Available Date: Aug 25, 2019
• Journal: ACS Omega
• Electronic ISSN: 2470-1343
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 3
• Issue: 8
• Pages: 8903-8912
• DOI: https://doi.org/10.1021/acsomega.8b00809
• Keywords: Baker’s yeast, biotransformation, codeine, CYP2D6 variants, morphine, SNPs.
• Public URL: https://uwe-repository.worktribe.com/output/862217
• Publisher URL: http://dx.doi.org/10.1021/acsomega.8b00809
• Related Public URLs: https://cdn-pubs.acs.org
• Additional Information: Additional Information : This is the author's accepted manuscript. The final published version is available here: http://dx.doi.org/10.1021/acsomega.8b00809.
• Contract Date: Aug 7, 2018