Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria.
We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.
Novikova
L A
LA
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia.
Nazarov
P A
PA
Saveliev
A S
AS
Drutsa
V L
VL
Sergeev
V N
VN
Miller
W L
WL
Luzikov
V N
VN
D000325:Adrenodoxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008928:Mitochondria / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011284:Pregnenolone / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000325:Adrenodoxin; D011284:Pregnenolone; D004247:DNA; D013250:Steroid Hydroxylases; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase