Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Feb;170(2):781–789. doi: 10.1128/jb.170.2.781-789.1988

Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination.

Y C Chen 1, O P Peoples 1, C T Walsh 1
PMCID: PMC210722  PMID: 3338974

Abstract

The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli.

Full text

PDF
781

Images in this article

Selected References

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

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Bigbee W. L., Vanderlaan M., Fong S. S., Jensen R. H. Monoclonal antibodies specific for the M- and N-forms of human glycophorin A. Mol Immunol. 1983 Dec;20(12):1353–1362. doi: 10.1016/0161-5890(83)90166-9. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Donoghue N. A., Norris D. B., Trudgill P. W. The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871. Eur J Biochem. 1976 Mar 16;63(1):175–192. doi: 10.1111/j.1432-1033.1976.tb10220.x. [DOI] [PubMed] [Google Scholar]
  6. Forster A. C., McInnes J. L., Skingle D. C., Symons R. H. Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. 1985 Feb 11;13(3):745–761. doi: 10.1093/nar/13.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Greer S., Perham R. N. Glutathione reductase from Escherichia coli: cloning and sequence analysis of the gene and relationship to other flavoprotein disulfide oxidoreductases. Biochemistry. 1986 May 6;25(9):2736–2742. doi: 10.1021/bi00357a069. [DOI] [PubMed] [Google Scholar]
  8. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Henriksen S. D. Moraxella, neisseria, branhamella, and acinetobacter. Annu Rev Microbiol. 1976;30:63–83. doi: 10.1146/annurev.mi.30.100176.000431. [DOI] [PubMed] [Google Scholar]
  10. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  11. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaplan J. B., Goncharoff P., Seibold A. M., Nichols B. P. Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster. Mol Biol Evol. 1984 Nov;1(6):456–472. doi: 10.1093/oxfordjournals.molbev.a040331. [DOI] [PubMed] [Google Scholar]
  13. Krauth-Siegel R. L., Blatterspiel R., Saleh M., Schiltz E., Schirmer R. H., Untucht-Grau R. Glutathione reductase from human erythrocytes. The sequences of the NADPH domain and of the interface domain. Eur J Biochem. 1982 Jan;121(2):259–267. doi: 10.1111/j.1432-1033.1982.tb05780.x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  16. Rossmann M. G., Moras D., Olsen K. W. Chemical and biological evolution of nucleotide-binding protein. Nature. 1974 Jul 19;250(463):194–199. doi: 10.1038/250194a0. [DOI] [PubMed] [Google Scholar]
  17. Ryerson C. C., Ballou D. P., Walsh C. Mechanistic studies on cyclohexanone oxygenase. Biochemistry. 1982 May 25;21(11):2644–2655. doi: 10.1021/bi00540a011. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Smith G. E., Summers M. D. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. doi: 10.1016/0003-2697(80)90019-6. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Staden R. A new computer method for the storage and manipulation of DNA gel reading data. Nucleic Acids Res. 1980 Aug 25;8(16):3673–3694. doi: 10.1093/nar/8.16.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Staden R. A strategy of DNA sequencing employing computer programs. Nucleic Acids Res. 1979 Jun 11;6(7):2601–2610. doi: 10.1093/nar/6.7.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Staden R., McLachlan A. D. Codon preference and its use in identifying protein coding regions in long DNA sequences. Nucleic Acids Res. 1982 Jan 11;10(1):141–156. doi: 10.1093/nar/10.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Wierenga R. K., Drenth J., Schulz G. E. Comparison of the three-dimensional protein and nucleotide structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase with the FAD- as well as NADPH-binding domains of glutathione reductase. J Mol Biol. 1983 Jul 5;167(3):725–739. doi: 10.1016/s0022-2836(83)80106-5. [DOI] [PubMed] [Google Scholar]
  26. Wierenga R. K., Terpstra P., Hol W. G. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. doi: 10.1016/0022-2836(86)90409-2. [DOI] [PubMed] [Google Scholar]
  27. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES