J. Biochem, 1992, Vol. 112, No. 1 155-162
© 1992 Japanese Biochemical Society
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Further Studies on Chimeric P450 2C2/2C14 Having Testosterone 16ß-Hydroxylase Activity Which Is Absent in the Parental P450s1
*Institute for Protein Research, Osaka University Suita, Osaka 565
**Department of Veterinary Science, University of Osaka Prefecture Sakai Osaka 593
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 16ß-hydroxylase activity, which is not seen in either of the parental P450s, in addition to higher activities of laurate (
-1)-hydroxylation and benzphetarnine 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 16ß-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 16ß- and laurate (-1)-hydroxylase activities. Further replacing the region 90-125 with that of P450 2C14 resulted in disappearance of the 16ß-hydroxylase activity and profound decrease in the (-1)-hydroxylase activity. Testosterone 16ß-hydroxylation was inhibited by laurate and laurate (-1)-hydroxylation by testosterone. These findings indicate that testosterone 16ß-hydroxylase activity is produced only when the specific segments of P450 2C2 and P450 2C14 are combined, and that at least the 90-125 region of P450 2C2 participates in both testosterone 16ß-hydroxylase and laurate (-1)-hydroxylase activities. To analyze the increase in the laurate hydroxylase activity on replacing the carboxy-terminal segment of P450 2C2, the rate of P450 reduction by NADPH and the ratio of NADPH oxidation to the product formation in the reconstituted hydroxylase system were measured. From the results, the possibility is discussed that a local conformational change is induced by replacing the carboxy-terminal segment of P450 2C2 with that of P450 2C14, resulting in improvement of the interaction between the oxygenated P450 intermediate and the electron-supplying system in the rate-limiting step of the overall hydroxylase reaction.
1This work was supported in part by a Grant-in-Aid for Scientific Research (63635001) from the Ministry of Education, Science and Culture of Japan. Individual forms of P450 are designated according to the nomenclature recommended by Nebert et al. (1).
Present address: 2Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo 657.
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