Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells.
Three cDNAs for chimeras between cytochrome P-450s (pHP3 and pHP2-1) were constructed and inserted between the alcohol dehydrogenase promoter and terminator regions of the yeast expression vector pAAH5 to form expression plasmids, pAH3P2, pAH3E2, and pAH3A2. pAH3P2 contained the entire coding sequence of cytochrome P-450 (pHP2-1) except for the 3rd, the 8th, the 36th, and the 42nd residues of the total of 490 amino acids. Nucleotide sequences of pAH3P2 were replaced with those of cytochrome P-450 (pHP3) in the region coding for the NH2-terminal 210 and 262 amino acid residues to yield pAH3E2 and pAH3A2, respectively. The three expression plasmids were introduced into Saccharomyces cerevisiae AH22 cells and cytochrome P-450 s (3P2, 3E2, and 3A2) were purified from the microsomal fractions of the transformed yeast cells. In the oxidized state either of the cytochromes exhibited a low- and high-spin mixed-type spectrum of cytochrome P-450. The reduced CO complex of the cytochromes showed a Soret absorption maximum at 450 nm. When laurate or caprate was added to ferric cytochrome P-450 s (3P2 and 3E2), the spectrum was converted to that of the typical high-spin type, indicating the binding of the fatty acids to the substrate site of the cytochromes. On the other hand, the addition of the fatty acids to ferric cytochrome P-450 (3A2) induced no spectral change. Only chemicals having a carboxyl group caused such spectral conversion of cytochrome P-450 (3P2) among dodecyl compounds examined.(ABSTRACT TRUNCATED AT 250 WORDS)
Imai
Y
Y
Institute for Protein Research, Osaka University.
D000595:Amino Acid Sequence; D000818:Animals; D002678:Chimera; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A