Further studies on chimeric P450 2C2/2C14 having testosterone 16 beta-hydroxylase activity which is absent in the parental P450s.
cDNAs for various chimeras between P450 2C2, P450 2C14, P450 2B5, and P450 2E1 were constructed, the chimeric P450s were expressed in yeast cells, and their catalytic activities were compared in the reconstituted system containing partially purified P450 preparations. The chimera P450(2Hc3), consisting of the 462 amino-terminal residues of P450 2C2 and the remaining 28 residues of P450 2C14, had testosterone 16 beta-hydroxylase activity, which is not seen in either of the parental P450s, in addition to higher activities of laurate (omega-1)-hydroxylation and benzphetamine N-demethylation than the parental P450s [Uno, T. et al. (1990) Biochem. Biophys. Res. Commun. 167, 498-503]. When either of the segments from P450 2C2 and P450 2C14 in this chimera was replaced with the corresponding sequences of P450 2E1 or when the 35 carboxy-terminal residues of P450(2Hc3) were replaced with those of P450 2B5, the 16 beta-hydroxylase activity disappeared. When the 262 amino-terminal residues, except for residues 90-125 (region 90-125), of P450(2Hc3) were replaced with those of P450 2C14, the resulting chimera retained both testosterone 16 beta- and laurate (omega-1)-hydroxylase activities. Further replacing the region 90-125 with that of P450 2C14 resulted in disappearance of the 16 beta-hydroxylase activity and profound decrease in the (omega-1)-hydroxylase activity. Testosterone 16 beta-hydroxylation was inhibited by laurate and laurate (omega-1)-hydroxylation by testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)
Uno
T
T
Institute for Protein Research, Osaka University.
Imai
Y
Y
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase