Chimeras of the human cytochrome P450 1A family produced in yeast. Accumulation in microsomal membranes, enzyme kinetics and stability.
An expression library of hybrid cDNAs was constructed in vivo by homeologous recombination in yeast between human P450 1A1 and P450 1A2 sequences. Two clones exhibiting highly enhanced monooxygenase activities in vivo were selected. Chimera S12 includes the 88 N-terminal residues of P450 1A1 fused to the complementary part of the P450 1A2 sequence. Chimera S71 derives from P450 1A1 by the substitution of the 36 C-terminal amino acid residues by the corresponding 38 residues of the 1A2 sequence. Biochemical analysis on microsomal fractions indicated that S12 and S71 have the same substrate specificities as 1A2 and 1A1, respectively. The observed increase in the in vivo monooxygenase activity is related to a ninefold increase in the microsomal S12 content as compared to the 1A2 content. In contrast, the expression level of S71 is slightly reduced but its turnover numbers are increased as compared to 1A1. The folding stability of chimeric P450 enzymes was evaluated by thermal and chaotropic agent denaturation. No difference was found between S12 and 1A2, but S71 appeared slightly less stable than 1A1. In vivo experiments indicated that S12 mRNA accumulation and stability are quite similar to the stability of parental 1A2 and, for both chimeras and parental enzymes, the protein half-lives are longer than the cell doubling time. The surprising accumulation of chimera S12 in the microsomal membrane is discussed in terms of the relationship of protein folding with transport to the endoplasmic reticulum membrane and the apparent expression levels of human P450 enzymes produced in yeast.
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Université Pierre-et-Marie Curie, Gif-sur-Yvette, France.
A
A
Bellamine
J C
JC
Gautier
P
P
Urban
D
D
Pompon
D000595:Amino Acid Sequence; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2