Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Nov;86(21):8262–8265. doi: 10.1073/pnas.86.21.8262

Hybrid cytochromes P-450 identify a substrate binding domain in P-450IIC5 and P-450IIC4.

T Kronbach 1, T M Larabee 1, E F Johnson 1
PMCID: PMC298260  PMID: 2813390

Abstract

The cytochrome P-450 superfamily of enzymes catalyzes the oxidative metabolism of innumerable lipophilic compounds (e.g., drugs, carcinogens, steroids). Although the three-dimensional structure of a soluble bacterial P-450 (P-450cam) has been solved, little is known about the structures of the membrane-bound mammalian P-450s. Thus, the structural features of these enzymes that determine their multisubstrate specificity are unknown. In this report, we identify a segment of the primary structure of the structurally similar but functionally distinct cytochromes P-450IIC5 and P-450IIC4, which determines the apparent affinity of these cytochromes for the conversion of progesterone into the mineralocorticoid deoxycorticosterone. P-450IIC5 exhibits a greater than 10-fold lower apparent Km than P-450IIC4 for progesterone 21-hydroxylation. Chimeric cDNAs were constructed and expressed in COS-1 cells, which encode hybrids between these enzymes. The hybrid enzymes were assayed for catalytic activity and compared to the parental proteins. A segment of P-450IIC5 was identified that conferred the lower Km of P-450IIC5 to P-450IIC4. Sequential reduction of the length of the exchanged segments led to a hybrid enzyme with a high affinity derived largely from P-450IIC4, which contains three amino acid residues derived from P-450IIC5 clustered between positions 113 and 118. This suggests that this region is part of a substrate binding domain. This region maps by alignment of amino acid sequences to a residue of P-450cam, which has been implicated in substrate binding, suggesting that these segments of the primary structure serve a similar functional role in these two distantly related proteins.

Full text

PDF
8262

Images in this article

8263Image
on p.8263
8263Image
on p.8263

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atkins W. M., Sligar S. G. The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis. J Biol Chem. 1988 Dec 15;263(35):18842–18849. [PubMed] [Google Scholar]
  2. Cullen B. R. Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 1987;152:684–704. doi: 10.1016/0076-6879(87)52074-2. [DOI] [PubMed] [Google Scholar]
  3. Dieter H. H., Muller-Eberhard U., Johnson E. F. Identification of rabbit microsomal cytochrome P-450 isozyme, form 1, as a hepatic progesterone 21-hydroxylase. Biochem Biophys Res Commun. 1982 Mar 30;105(2):515–520. doi: 10.1016/0006-291x(82)91465-6. [DOI] [PubMed] [Google Scholar]
  4. Dieter H. H., Muller-Eberhard U., Johnson E. F. Rabbit hepatic progesterone 21-hydroxylase exhibits a bimodal distribution of activity. Science. 1982 Aug 20;217(4561):741–743. doi: 10.1126/science.6808664. [DOI] [PubMed] [Google Scholar]
  5. Garger S. J., Griffith O. M., Grill L. K. Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient. Biochem Biophys Res Commun. 1983 Dec 28;117(3):835–842. doi: 10.1016/0006-291x(83)91672-8. [DOI] [PubMed] [Google Scholar]
  6. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Imai Y. Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells. J Biochem. 1988 Jan;103(1):143–148. doi: 10.1093/oxfordjournals.jbchem.a122220. [DOI] [PubMed] [Google Scholar]
  8. Johnson E. F., Barnes H. J., Griffin K. J., Okino S., Tukey R. H. Characterization of a second gene product related to rabbit cytochrome P-450 1. J Biol Chem. 1987 Apr 25;262(12):5918–5923. [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Nebert D. W., Nelson D. R., Adesnik M., Coon M. J., Estabrook R. W., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F., Kemper B. The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci. DNA. 1989 Jan-Feb;8(1):1–13. doi: 10.1089/dna.1.1989.8.1. [DOI] [PubMed] [Google Scholar]
  11. Pompon D. cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6. Eur J Biochem. 1988 Nov 1;177(2):285–293. doi: 10.1111/j.1432-1033.1988.tb14375.x. [DOI] [PubMed] [Google Scholar]
  12. Poulos T. L., Finzel B. C., Howard A. J. High-resolution crystal structure of cytochrome P450cam. J Mol Biol. 1987 Jun 5;195(3):687–700. doi: 10.1016/0022-2836(87)90190-2. [DOI] [PubMed] [Google Scholar]
  13. Reubi I., Griffin K. J., Raucy J. L., Johnson E. F. Three monoclonal antibodies to rabbit microsomal cytochrome P-450 1 recognize distinct epitopes that are shared to different degrees among other electrophoretic types of cytochrome P-450. J Biol Chem. 1984 May 10;259(9):5887–5892. [PubMed] [Google Scholar]
  14. Sakaki T., Shibata M., Yabusaki Y., Ohkawa H. Expression in Saccharomyces cerevisiae of chimeric cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d. DNA. 1987 Feb;6(1):31–39. doi: 10.1089/dna.1987.6.31. [DOI] [PubMed] [Google Scholar]
  15. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  16. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tukey R. H., Okino S., Barnes H., Griffin K. J., Johnson E. F. Multiple gene-like sequences related to the rabbit hepatic progesterone 21-hydroxylase cytochrome P-450 1. J Biol Chem. 1985 Oct 25;260(24):13347–13354. [PubMed] [Google Scholar]
  20. Zuber M. X., Simpson E. R., Waterman M. R. Expression of bovine 17 alpha-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells. Science. 1986 Dec 5;234(4781):1258–1261. doi: 10.1126/science.3535074. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES