Abstract
The nucleotide sequence of cDNA for rabbit liver cytochrome P-450 (laurate (ω-1)-hydroxylase) was replaced with that for rabbit liver cytochrome P-450 (testosterone 16α-hydroxylase) in various regions coding for the amino acid sequence between residues 43 and 261. Six chimeric cDNAs thus constructed were cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. Chimeric P-450s synthesized in the transformed yeast cells were purified partially and their catalytic and spectral properties were examined and compared with those of the chimeric P-450 which is considered to possess the same catalytic properties as the wild-type P-450. In the oxidized state the chimeric P-450s exhibited a low- and high-spin mixed-type absorption spectrum of cytochrome P-450 and the spectrum was converted to a typical high-spin type on addition of laurate or caprate, indicating the binding-of the fatty acids to the substrate site of the chimeric P-450s. However, the affinities of the fatty acids for the chimeras devoid of the sequence of P-450 (laurate (ω-1)-hydroxylase) in either of the regions spanning residues 90-125 and 210-261 were 10 to 20 times lower than those for the chimeras containing the sequence of the wild-type P-450 in both regions. The latter chimeras have about the same affinities as the chimera which is essentially the wild-type P-450. In the reconstituted system containing purified enzymes, the chimeras containing the sequence of the wild-type P-450 in both regions catalyzed (ω-1)-hydroxylation of the fatty acids, but their activities were 51-67% of that of the chimera which is essentially the wild-type P-450. The chimera which contains the sequence of the wild-type P-450 in the region covering residues 90-125 but not residues 210-261 was about one-tenth as active as the chimera which is essentially the wild-type P-450. On the other hand, the chimeras devoid of the sequence of the wild-type P-450 in the region spanning residues 90-125 were completely devoid of the hydroxylase activities. These results indicate that two segments of P-450 (laurate (ω-1)-hydroxylase) covering residues 90-125 and 210-262 constitute the substrate binding sites and cooperate to fix the fatty acid at an appropriate position on the P-450 molecule, and the former segment is essential to the hydroxylase activity. The reduced CO complex of the chimeric P-450s showed the typical absorption spectrum of cytochrome P-450. However, the CO complexes of the chimeras devoid of the sequence of the wild-type P-450 covering residues 90-125 were much more unstable to denaturation than those containing this sequence, suggesting that this sequence protects the conformation of the chimeric P-450s in the reduced state against denaturation.
