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  • Title: Genetically engineered P450 monooxygenases: construction of bovine P450c17/yeast reductase fused enzymes.
    Author: Shibata M, Sakaki T, Yabusaki Y, Murakami H, Ohkawa H.
    Journal: DNA Cell Biol; 1990; 9(1):27-36. PubMed ID: 2180429.
    Abstract:
    Seven P450/reductase fused enzymes were produced in Saccharomyces cerevisiae by expressing fused cDNAs consisting of bovine cytochrome P450c17 (P450c17) and yeast NADPH-cytochrome P450 reductase (reductase). These fused enzymes differed in the length and amino acid sequence of the hinge region between the P450 and reductase moieties. Expression of the fused constructs under the control of the yeast alcohol dehydrogenase I promoter and terminator of expression vector pAAH5 in S. cerevisiae AH22 cells resulted in the production of about 2-8 X 10(4) molecules per cell of the seven corresponding fused enzymes. Six of the fused enzymes incorporated a protoheme, as confirmed by reduced CO-difference spectra. Recombinant yeast strains producing each of the fused hemoproteins showed P450c17-dependent 17 alpha-hydroxylase activity toward progesterone. The most active fused enzyme, delta N23FE, which lacked the amino-terminal 23 amino acids of the reductase, showed about 10 times higher 17 alpha-hydroxylase activity than bovine P450c17, although the fused enzyme (delta N23FE)' with an amino acid sequence in the hinge region different from delta N23FE was less active than delta N23FE. The fused enzyme delta N0FE, consisting of P450c17 and whole reductase, showed about 1.8 times higher activity than bovine P450c17. No activity was found with delta N84FE lacking the amino-terminal 84 amino acids of the reductase moiety. P450c17-dependent C17,(20)-lyase activity toward 17 alpha-hydroxyprogesterone was detected to lesser extents in the recombinant yeast. Fused bovine P450c17/yeast reductase enzymes show enhanced 17 alpha-hydroxylase activity, and the length and amino acid sequence in the hinge region between the P450c17 and yeast reductase moieties can be important for efficient intramolecular electron transfer in the fused enzymes.
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