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. 1986 Oct 24;14(20):8061–8071. doi: 10.1093/nar/14.20.8061

Cloning and sequencing of chloroperoxidase cDNA.

G H Fang, P Kenigsberg, M J Axley, M Nuell, L P Hager
PMCID: PMC311834  PMID: 3774552

Abstract

An oligod-d(T) 12-18 primed cDNA library has been prepared from Caldariomyces fumago mRNA. A clone containing a full-length insert was sequenced on the supercoiled plasmid, pBR322. The complete primary sequence of chloroperoxidase has been derived. We have also determined about 73% of the peptide sequence by amino acid sequencing. The DNA sequence data matches all of the available known peptide sequences. The mature polypeptide contains 300 amino acids having a combined molecular weight of 32,974 daltons. A putative signal peptide of 21 amino acids is proposed from DNA sequence data. The chloroperoxidase gene encodes three potential glycosylation sites recognized as Asn-X-Thr/Ser sequences. Three cysteine residues are found in the protein sequence. A small region around Cys87 bears a minimal homology to the active site of cytochrome P450cam. No other heme protein homologues can be detected. We propose that Cys87 serves as a thiolate ligand to the iron of heme prosthetic group. A rare arginine codon, AGG, is used three times out of twelve in contrast to the very infrequent use of this codon in E. coli or yeast.

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Selected References

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

  1. Bangcharoenpaurpong O., Champion P. M., Hall K. S., Hager L. P. Resonance Raman studies of isotopically labeled chloroperoxidase. Biochemistry. 1986 May 6;25(9):2374–2378. doi: 10.1021/bi00357a011. [DOI] [PubMed] [Google Scholar]
  2. Brown F. S., Hager L. P. Chloroperoxidase. IV. Evidence for an ionic electrophilic substitution mechanism. J Am Chem Soc. 1967 Feb 1;89(3):719–720. doi: 10.1021/ja00979a061. [DOI] [PubMed] [Google Scholar]
  3. Champion P. M., Chiang R., Münck E., Debrunner P., Hager L. P. Mössbauer investigations of high-spin ferrous heme proteins. II. Chloroperoxidase, horseradish peroxidase, and hemoglobin. Biochemistry. 1975 Sep 23;14(19):4159–4166. doi: 10.1021/bi00690a002. [DOI] [PubMed] [Google Scholar]
  4. Champion P. M., Münck E., Debrunner P. G., Hollenberg P. F., Hager L. P. Mössbauer investigations of chloroperoxidase and its halide complexes. Biochemistry. 1973 Jan 30;12(3):426–435. doi: 10.1021/bi00727a011. [DOI] [PubMed] [Google Scholar]
  5. Chiang R., Makino R., Spomer W. E., Hager L. P. Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups. Biochemistry. 1975 Sep 23;14(19):4166–4171. doi: 10.1021/bi00690a003. [DOI] [PubMed] [Google Scholar]
  6. Dawson J. H., Trudell J. R., Barth G., Linder R. E., Bunnenberg E., Djerassi C., Chiang R., Hager L. P. Letter: Chloroperoxidase. Evidence for P-450 type heme environment from magnetic circular dichroism spectroscopy. J Am Chem Soc. 1976 Jun 9;98(12):3709–3710. doi: 10.1021/ja00428a055. [DOI] [PubMed] [Google Scholar]
  7. Deng G., Wu R. Terminal transferase: use of the tailing of DNA and for in vitro mutagenesis. Methods Enzymol. 1983;100:96–116. doi: 10.1016/0076-6879(83)00047-6. [DOI] [PubMed] [Google Scholar]
  8. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  10. Hager L. P., Morris D. R., Brown F. S., Eberwein H. Chloroperoxidase. II. Utilization of halogen anions. J Biol Chem. 1966 Apr 25;241(8):1769–1777. [PubMed] [Google Scholar]
  11. Hollenberg P. F., Hager L. P., Blumberg W. E., Peisach J. An electron paramagnetic resonance study of the high and low spin forms of chloroperoxidase. J Biol Chem. 1980 May 25;255(10):4801–4807. [PubMed] [Google Scholar]
  12. Hollenberg P. F., Hager L. P. The P-450 nature of the carbon monoxide complex of ferrous chloroperoxidase. J Biol Chem. 1973 Apr 10;248(7):2630–2633. [PubMed] [Google Scholar]
  13. Libby R. D., Thomas J. A., Kaiser L. W., Hager L. P. Chloroperoxidase halogenation reactions. Chemical versus enzymic halogenating intermediates. J Biol Chem. 1982 May 10;257(9):5030–5037. [PubMed] [Google Scholar]
  14. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  15. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  17. Morris D. R., Hager L. P. Chloroperoxidase. I. Isolation and properties of the crystalline glycoprotein. J Biol Chem. 1966 Apr 25;241(8):1763–1768. [PubMed] [Google Scholar]
  18. Remba R. D., Champion P. M., Fitchen D. B., Chiang R., Hager L. P. Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation. Biochemistry. 1979 May 29;18(11):2280–2290. doi: 10.1021/bi00578a023. [DOI] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sono M., Dawson J. H., Hager L. P. The generation of a hyperporphyrin spectrum upon thiol binding to ferric chloroperoxidase. Further evidence of endogenous thiolate ligation to the ferric enzyme. J Biol Chem. 1984 Nov 10;259(21):13209–13216. [PubMed] [Google Scholar]
  21. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  22. Tan Z. K., Ikuta S., Huang T., Dugaiczyk A., Itakura K. Solid-phase synthesis of polynucleotides. VIII: A simplified synthesis of oligodeoxyribonucleotides. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):383–391. doi: 10.1101/sqb.1983.047.01.045. [DOI] [PubMed] [Google Scholar]
  23. Thomas J. A., Morris D. R., Hager L. P. Chloroperoxidase. VII. Classical peroxidatic, catalatic, and halogenating forms of the enzyme. J Biol Chem. 1970 Jun;245(12):3129–3134. [PubMed] [Google Scholar]

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