Expression in yeast of three allelic cDNAs coding for human liver P-450 3A4. Different stabilities, binding properties and catalytic activities of the yeast-produced enzymes.
Three natural allelic cDNAs coding for P-450 3A4, the major form in human liver, namely NF25, NF10 and hPCN1, have been expressed in Saccharomyces cerevisiae. NF25 and hPCN1 were functionally expressed in yeast microsomes, yielding proteins with an absorption maximum at 448 nm in the CO-reduced difference spectrum. Some catalytic activities and substrate binding properties of P-450 NF25 and P-450 hPCN1 in yeast microsomes have been compared; no striking difference was found, showing that the two point substitutions between their amino-acid sequences (Trp392 and Thr431 in P-450 NF25 are replaced by Val392 and Ile431 in P-450 hPCN1) have no significant effect on the functional properties of these two variants. By contrast, P-450 NF10, which differs from P-450 NF25 by a one-amino-acid deletion (Ile224 replacing Thr224-Val225), was produced as a denatured form, as revealed by an absorption maximum at 420 nm, and was not catalytically active. This suggests that the deletion prevents the correct folding of the protein. The results of this study show that P-450 NF25 and P-450 hPCN1 are two roughly equivalent, functionally active variants of P-450 3A4, but that P-450 NF10 is a defective, unstable gene product that could arise from an alternative mRNA splicing. This could contribute to the large variations reported for nifedipine oxidation, a typical P-450 3A4 activity, in human liver.
Peyronneau
M A
MA
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS URA 400, Université Paris 5, France.
Renaud
J P
JP
Jaouen
M
M
Urban
P
P
Cullin
C
C
Pompon
D
D
Mansuy
D
D
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D004795:Enzyme Stability; D006801:Humans; D008099:Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A