Functional properties of CYP2D6 1 (wild-type) and CYP2D6 7 (His324Pro) expressed by recombinant baculovirus in insect cells.
The debrisoquine/sparteine or CYP2D6 genetic polymorphism of drug oxidation is a common cause for interindividual variability in drug response. We recently identified a mutant allele, designated CYP2D6-E or CYP2D6*7, which is associated with the poor metabolizer phenotype and occurs in Caucasian populations with a frequency of about 1%. In contrast to other loss-of-function alleles, a full length protein with a single amino acid substitution. His324Pro, is encoded by the CYP2D6*7 allele. To functionally analyze this mutant protein form of CYP2D6, recombinant baculoviruses were constructed to express the CYP2D6 cDNA. Up to 0.33 nmol of spectrally detected P450/mg of cell protein were produced in Spodoptera frugiperda cells, whereas Trichoplusia ni 5B1-4 cells reproducibly produced 0.8 nmol/mg (4% of total cell protein). Insect cell membranes were functionally characterized with cumene hydroperoxide or after reconstitution with purified rat NADPH:cytochrome P450 reductase. Km values for the substrates bufuralol and sparteine and other enzymatic properties were almost identical to those of human liver microsomes. The H324P mutation was introduced into the cDNA by site-directed mutagenesis and recombinant baculovirus was obtained. Expression under a variety of conditions demonstrated that mutant protein amounts comparable to the wild-type enzyme were produced. However, no spectrally detectable P450 was formed and no catalytic activity was detected. Furthermore, in contrast to the wild-type protein the mutant protein was almost exclusively located in a detergent-insoluble insect cell fraction. These results demonstrate that the H324P mutation is responsible for the in vivo poor metabolizer phenotype associated with the CYP2D6*7 allele by preventing normal protein folding and heme incorporation.
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Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart, Germany.
Eichelbaum
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Haubruck
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Zanger
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D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D002478:Cells, Cultured; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D007313:Insecta / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D016296:Mutagenesis; D010641:Phenotype; D011110:Polymorphism, Genetic / Q000235:genetics; D051381:Rats; D018411:Spodoptera / Q000378:metabolism
D007527:Isoenzymes; D019389:Cytochrome P-450 CYP2D6