Skip to main content

Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. DS002 and Cloning, Sequencing of Partial catA Gene

Indian Journal of Microbiology volume 51, pages 312–318 (2011)Cite this article

Abstract

Catechol 1,2-dioxygenase (C12O) was purified to electrophoretic homogeneity from Acinetobacter sp. DS002. The pure enzyme appears to be a homodimer with a molecular mass of 66 kDa. The apparent Km and Vmax for intradiol cleavage of catechol were 1.58 μM and 2 units per mg of protein respectively. Unlike other C12Os reported in the literature, the catechol 1,2-dioxygenase of Acinetobacter showed neither intradiol nor extradiol cleavage activity when substituted catechols were used as substrates. However, it has shown mild intradiol cleavage activity when benzenetriol was used as substrate. As determined by two dimensional electrophoresis (2DE) followed MALDI-TOF/TOF analyses and gel permeation chromatography, no isoforms of C12O was observed in Acinetobacter sp. DS002. Further, the C12O was seen only in cultures grown in benzoate and it was completely absent in succinate grown cultures. Based on the sequence information obtained from MS/MS data, degenerate primers were designed to amplify catA gene from the genomic DNA of Acinetobacter sp. DS002. The sequence of the PCR amplicon and deduced amino acid sequence showed 97% similarity with a catA gene of Acinetobacter baumannii AYE (YP_001713609).

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Warhurst M, Fewson CA (1994) Biotransformations catalyzed by the genus Rhodococcus. Crit Rev Biotechnol 14:29–73

    PubMed  Article  CAS  Google Scholar 

  2. Bouwer EJ, Zehnder AJB (1993) Bioremediation of organic compounds—putting microbial metabolism to work. Trends Biotechnol 11:360–367

    PubMed  Article  CAS  Google Scholar 

  3. Williams PA, Sayer JR (1994) The evolution of pathways for aromatic hydrocarbon oxidation in Pseudomonas. Biodegradation 5:195–217

    PubMed  Article  CAS  Google Scholar 

  4. Houghton JE, Shanley MS (1994) Catabolic potential of Pseudomonads: a regulatory perspective. In: Chaudhry GR (ed) Biological degradation and bioremediation of toxic chemicals. Dioscorides Press, Portland, pp 11–32

    Google Scholar 

  5. Johnson BF, Stanier RY (1971) Dissimilation of aromatic compounds by Alcaligenes eutrophus. J Bacteriol 107:468–475

    PubMed  CAS  Google Scholar 

  6. Hayaishi O, Katagiri M, Rothberg S (1957) Studies on oxygenases: pyrocatechase. J Biol Chem 229:905–9207

    PubMed  CAS  Google Scholar 

  7. Patel RN, Hou CT, Felix A, Lillard MO (1976) Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties. J Bacteriol 127:536–544

    PubMed  CAS  Google Scholar 

  8. Aoki K, Konohana T, Shinke R, Nishira H (1984) Purification and characterization of catechol 1,2-dioxygenase from aniline-assimilating Rhodococcus erythropolis AN- 13. Agric Biol Chem 48:2087–2095

    CAS  Google Scholar 

  9. Aoki K, Konohana T, Shinke R, Nishira H (1984) Two catechol 1,2-dioxygenases from aniline-assimilating bacterium, Frateuria species ANA-18. Agric Biol Chem 48:2097–2104

    CAS  Google Scholar 

  10. Dorn E, Knackmuss HJ (1978) Chemical structure and biodegradability of halogenated aromatic compounds: two catechol 1,2-dioxygenases from a 3- chlorobenzoate grown pseudomonad. Biochem J 174:73–84

    PubMed  CAS  Google Scholar 

  11. Fujiwara M, Golovleva LA, Saeki Y, Nozaki M, Hayaishi O (1975) Extradiol cleavage of 3-substituted catechols by an intradiol dioxygenase, pyrocatechase, from a pseudomonad. J Biol Chem 250:4848–4855

    PubMed  CAS  Google Scholar 

  12. Suresh BP, Purushotham G, Pijari AB, Krovidi RK, Baru R, Yanamandra M, Merrick M, Siddavattam D (2006) Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2–hydroxylase in a Gram negative Serratia sp. strain DS001. Appl Microbiol Biotechnol 73:1452–1462

    Google Scholar 

  13. Briganti Fabrizio, Pessione Enrica, Giunta Carlo, Scozzafava Andrea (1997) Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens. FEBS Lett 416:61–6414

    PubMed  Article  CAS  Google Scholar 

  14. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Article  CAS  Google Scholar 

  15. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 772:248–254

    Article  Google Scholar 

  16. Stothard P (2000) The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102–1104

    PubMed  CAS  Google Scholar 

  17. Strachan PD, Freer AA, Fewson CA (1998) Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene. Biochem J 333(3):741–747

    PubMed  CAS  Google Scholar 

  18. Ridder L, Briganti F, Boersma MG, Boeren S, Vis EH, Scozzafava A, Veeger C, Rietjens IMCM (1998) Quantitative structure/activity relationship for the rate of conversion of C4-substituted catechols by catechol 1,2-dioxygenase from Pseudomonas putida (arvilla) C1. Eur J Biochem 257:92–100

    PubMed  Article  CAS  Google Scholar 

  19. Wang C, You S, Wang S (2006) Purification and characterization of a novel catechol 1,2-dioxygenase from Pseudomonas aeruginosa with benzoic acid as a carbon source. Proc Biochem 41:1594–1601

    Article  CAS  Google Scholar 

  20. Briganti F, Pessione E, Giunta C, Mazzoli R, Scozzafava A (2000) Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate grown cells of Acinetobacter radioresistens. J Protein Chem 19:709–716

    PubMed  Article  CAS  Google Scholar 

  21. Murakami S, Wang CL, Naito A, Shinke R, Aoki K (1998) Purification and characterization of four catechol 1,2-dioxygenase isozymes from the benzamide assimilating bacterium Arthrobacter species BA-5–17. Microbiol Res 153(2):163–171

    PubMed  CAS  Google Scholar 

  22. Nakai C, Kagamiyama H, Saeki Y, Nozaki M (1979) Nonidentical subunits of pyrocatechase from Pseudomonas arvilla C-1. Arch Biochem Biophys 195:12–22

    PubMed  Article  CAS  Google Scholar 

  23. Canovas JL, Stanier RY (1967) Regulation of the enzymes of the β-ketoadipate pathway in Moraxella calcoaceticus. Eur J Biochem 1:289–300

    PubMed  Article  CAS  Google Scholar 

  24. Ornston LN (1971) Regulation of catabolic pathways in Pseudomonas. Bacteriol Rev 35:87–116

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

PEVP is a recipient of Senior Research Fellowship (SRF) from Council of Scientific and Industrial Research (CSIR), New Delhi. Research work in the laboratory of DS is supported by Dept. of Science and Technology (DST), Govt. of India and CSIR, New Delhi.

Author information

Affiliations

  1. Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India

    Emmanuel Vijay Paul Pandeeti & Dayananda Siddavattam

Authors
  1. Emmanuel Vijay Paul Pandeeti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dayananda Siddavattam
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dayananda Siddavattam.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pandeeti, E.V.P., Siddavattam, D. Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. DS002 and Cloning, Sequencing of Partial catA Gene. Indian J Microbiol 51, 312–318 (2011). https://doi.org/10.1007/s12088-011-0123-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12088-011-0123-4

Keywords

  • Biodegradation
  • Proteomics
  • Ortho pathway