Expression of four rat CYP2D isoforms in Saccharomyces cerevisiae and their catalytic specificity.
We cloned four cDNAs belonging to the CYP2D subfamily to express these enzymes in yeast cells and to compare their catalytic activities simultaneously. Three are believed to be alleles of CYP2D1, 2D2, and 2D3, respectively, based on high nucleotide sequence similarity, while CYP2D4 had both sequences of CYP2D4 and CYP2D18. Expression plasmids carrying CYP2D cDNAs were transformed into Saccharomyces cerevisiae. Typical P450 CO-difference spectra with absorbance maximum at 448 nm were recorded with microsomal preparations from the yeast cells expressing the four CYP2D forms. A catalytic study of these CYP2D forms was done with debrisoquine, bufuralol, and lidocaine. CYP2D2 had the highest debrisoquine 4-hydroxylation (2.2 nmol/min/nmol P450) activity, similar to that (2.2 nmol/min/nmol) of human CYP2D6 expressed in yeast cells. CYP2D3 had high lidocaine N-deethylation (43 nmol/min/nmol P450) activity, and both CYP2D3 and 2D2 exhibited high lidocaine 3-hydroxylation (2.4 and 1.6 nmol/min/nmol P450, respectively) activity. Bufuralol 1'-hydroxylation catalytic capabilities were comparable among the four isoforms. The activity of CYP2D1 was relatively low toward the three substrates (debrisoquine, 0.091; bufuralol, 1.5; lidocaine 3-hydroxylation, 0.019; lidocaine N-deethylation, 2.8 nmol/min/nmol P450). These findings indicate that debrisoquine, a typical substrate for CYP2D forms, was mainly metabolized by CYP2D2 but not CYP2D1 in rat liver and that the CYP2D forms have different substrate specificity.
Wan
J
J
Laboratory of Chemistry, Osaka City University Medical School, Japan.
Imaoka
S
S
Chow
T
T
Hiroi
T
T
Yabusaki
Y
Y
Funae
Y
Y
D000818:Animals; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D003647:Debrisoquin / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008012:Lidocaine / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D004983:Ethanolamines; D007527:Isoenzymes; D011994:Recombinant Proteins; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D008012:Lidocaine; D003647:Debrisoquin