Expression, Purification, Biochemical Characterization, and Comparative Function of Human Cytochrome P450 2D6.1, 2D6.2, 2D6.10, and 2D6.17 Allelic Isoforms

Abstract

Polymorphism at the cytochrome P450 2D6 (CYP2D6) locus is one of the most widely known causes of pharmacogenetic variability in humans. Our goal is to investigate the intrinsic enzymatic differences that exist among active CYP2D6 isoforms to test the hypothesis that these enzymatic differences are substrate-dependent. Active CYP2D6.1, 2, 10, and 17 holoenzymes were expressed in vitro and purified to a high degree of homogeneity as confirmed with SDS-polyacrylamide gel electrophoresis, CO-difference spectroscopy, and mass spectral analysis. Purified enzyme was reconstituted with lipid and cytochrome P450 reductase in a 2:1 ratio before kinetic analysis. The reaction rate for dextromethorphan (DXM) O-demethylation, DXM N-demethylation, codeine O-demethylation, and fluoxetine N-demethylation catalyzed by each of the variants was determined. The CYP2D6.10 enzyme was the most impaired, exhibiting an estimated enzyme efficiency (as V max /K m ) 50-fold lower for DXM O-demethylation and 100-fold lower for fluoxetine N-demethylation when compared with CYP2D6.1, whereas no measurable catalytic activity was observed for this variant toward codeine. The atypical DXM N-demethylation pathway catalyzed by this variant decreased only 2-fold in comparison. In the case of CYPD6.17, estimated clearances for each metabolite were decreased 6 to 33%. Likewise, the intrinsic clearance of CYP2D6.2 enzyme was consistently decreased for each reaction examined, indicating that the ultra-rapid metabolizer phenotype sometimes associated with this genotype is not a function of the underlying amino acid substitutions. Overall enzyme efficiencies for the metabolism of each substrate therefore decreased in the order of 2D6.1 Ͼ 2D6.2 Ͼ 2D6.17 Ͼ 2D6.10.Cytochrome P450 enzymes, a superfamily of heme-thiolate proteins, are found in all living organisms and are involved in the biotransformation of a diverse range of xenobiotics and endobiotics. Human P450 isoforms, which are mainly expressed in the liver, play a central role in drug metabolism. Variations in individual metabolism often result in unexpected toxicities because drug clearance is affected by a range of factors, including genetic variation, enzyme induction (activation), and inhibition of drug metabolism. Therefore, characterization of the P450 enzyme family has been of unceasing interest for the prediction and identification of drug metabolism and drug-drug interactions for discovery, development, and clinical therapy (Gonzalez and

CYP2D6 genetic variation in healthy adults and psychiatric African-American subjects: implications for clinical practice and genetic testing

“…33 The concept of allele substrate-dependent versus allele global depression in CYP2D6 function is easiest to visualize from the CYP2D6*17 allele perspective. The CYP2D6*17 allele has generally been characterized as an allele with diminished CYP2D6 activity, 24,25,[34][35][36] but there is some evidence that this classification is wrong. 35,33 The psychiatric patients evaluated in this study came from a larger group of patients prescribed risperidone in which the risperidone and 9-OH risperidone levels were determined.…”

“…The CYP2D6*17 allele has generally been characterized as an allele with diminished CYP2D6 activity, 24,25,[34][35][36] but there is some evidence that this classification is wrong. 35,33 The psychiatric patients evaluated in this study came from a larger group of patients prescribed risperidone in which the risperidone and 9-OH risperidone levels were determined. 37 The risperidone/9-OH ratio in patient plasma is dependent on CYP2D6 activity as 9-OH risperidone is formed by CYP2D6 metabolism of risperidone.…”

Expression and Functional Analysis of CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7 Isozymes

“…The plasmid pFB2D6*1 (Yu et al, 2002) containing wild-type CYP2D6.1 cDNA was used as a template to create other CYP2D6 allelic variants by using the QuickChange Multi-Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following the manufacturer's recommendations. The oligonucleotides for creation of CYP2D6*24, CYP2D6*26, and CYP2D6*27 and an extensive CYP2D6 mutant (CYP2D6m) were shown in the supplemental table.…”

“…CYP2D6 null alleles such as CYP2D6*3, CYP2D6*4, and CYP2D6*5 could result in a CYP2D6 protein that is unable to bind the substrate, a truncated apoprotein unable to bind heme, or simply no CYP2D6 protein among CYP2D6 poor metabolizers. In contrast, some CYP2D6 alleles including CYP2D6*10 and CYP2D6*17 do produce functional CYP2D6 enzymes, whereas the CYP2D6 allelic isoforms have reduced protein stability and/or drugmetabolizing capacity (Johansson et al, 1994;Yu et al, 2002;Shen et al, 2007). Whereas the enzyme functions of many CYP2D6 allelic variants, including CYP2D6*36, CYP2D6*56, CYP2D6*59, and CYP2D6*62 (Gaedigk et al, 2006;Li et al, 2006;Toscano et al, 2006;Klein et al, 2007), have been determined, the drug-metabolizing capacity of proteins corresponding to other nonsynonymous alleles such as CYP2D6*24 (2853AϾC; I297L), CYP2D6*26 (3277TϾC; I369T), and CYP2D6*27 (3853GϾA; E410K) (supplemental table) that were found in 0.1 to 0.3% of whites (Marez et al, 1997) remain unknown.…”

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity

“…24 The CYP2D6.10 enzyme has a deleterious P34S mutation abolishing the important PPGP sequence necessary for folding of P450 and is therefore very unstable, 24 but it has also a reduced affinity for the substrates. 31 Among Blacks CYP2D6*17 first described in 1996 32 is the major variant CYP2D6 allele. It encodes in addition to the two missense mutations seen in CYP2D6*2 also T107I, which apparently makes an altered active site structure.…”