In a previous environmental survey of a polluted area, the authors identified two catechol 2,3-dioxygenase (C23O) sequences predominant in environmental bacterial isolates mineralizing benzene and/or toluene and also in soil DNA extracts. In the present study, using information of stable operon arrangement and conserved gene sequences, the complete C23O ORFs of these two variants were cloned, sequenced and overexpressed. The variants differ in six nucleotide positions, and the putative protein sequences differ only by a single amino acid, Tyr or His, at position 218. Even though the three-dimensional model does not suggest a significant influence of such an amino acid substitution on enzyme function, the Tyr218 variant differed significantly from the His218 variant in lower turnover number and in lower apparent for catecholic substrates. These results are evidence of the importance for enzyme function of amino acids not directly influencing active site structure and prove the utility of recovering polymorphisms evolved and selected for special functions in natural ecosystems.


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  1. Andujar, E. & Santero, E.(2003). Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity. Microbiology 149, 1559–1567.[CrossRef] [Google Scholar]
  2. Beil, S., Mason, J. R., Timmis, K. N. & Pieper, D. H.(1998). Identification of chlorobenzene dioxygenase sequence elements involved in dechlorination of 1,2,4,5-tetrachlorobenzene. J Bacteriol 180, 5520–5528. [Google Scholar]
  3. Bradford, M. M.(1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef] [Google Scholar]
  4. Bugg, T. D. H. & Lin, G.(2001). Solving the riddle of the intradiol and extradiol catechol dioxygenases: how do enzymes control hydroperoxide rearrangements? Chem Commun 2001, 941–952. [Google Scholar]
  5. Cerdan, P., Wasserfallen, A., Rekik, M., Timmis, K. N. & Harayama, S.(1994). Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth. J Bacteriol 176, 6074–6081. [Google Scholar]
  6. Cerdan, P., Rekik, M. & Harayama, S.(1995). Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida. Eur J Biochem 229, 113–118.[CrossRef] [Google Scholar]
  7. Eltis, L. D. & Bolin, J. T.(1996). Evolutionary relationships among extradiol dioxygenases. J Bacteriol 178, 5930–5937. [Google Scholar]
  8. Eltis, L. D., Hofmann, B., Hecht, H. J., Lunsdorf, H. & Timmis, K. N.(1993). Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase. J Biol Chem 268, 2727–2732. [Google Scholar]
  9. Harayama, S., Rekik, M., Wasserfallen, A. & Bairoch, A.(1987). Evolutionary relationships between catabolic pathways for aromatics: conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids. Mol Gen Genet 210, 241–247.[CrossRef] [Google Scholar]
  10. Harwood, C. S. & Parales, R. E.(1996). The beta-ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50, 553–590.[CrossRef] [Google Scholar]
  11. Heiss, G., Stolz, A., Kuhm, A. E., Muller, C., Klein, J., Altenbuchner, J. & Knackmuss, H. J.(1995). Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6. J Bacteriol 177, 5865–5871. [Google Scholar]
  12. Hirose, J., Kimura, N., Suyama, A., Kobayashi, A., Hayashida, S. & Furukawa, K.(1994). Functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of Pseudomonas origin. FEMS Microbiol Lett 118, 273–277.[CrossRef] [Google Scholar]
  13. Hugo, N., Meyer, C., Armengaud, J., Gaillard, J., Timmis, K. N. & Jouanneau, Y.(2000). Characterization of three XylT-like [2Fe-2S] ferredoxins associated with catabolism of cresols or naphthalene: evidence for their involvement in catechol dioxygenase reactivation. J Bacteriol 182, 5580–5585.[CrossRef] [Google Scholar]
  14. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M.(1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47, 110–119.[CrossRef] [Google Scholar]
  15. Junca, H. & Pieper, D. H.(2003). Amplified functional DNA restriction analysis to determine catechol 2,3-dioxygenase gene diversity in soil bacteria. J Microbiol Methods 55, 697–708.[CrossRef] [Google Scholar]
  16. Junca, H. & Pieper, D. H.(2004). Functional gene diversity analysis in BTEX contaminated soils by means of PCR-SSCP DNA fingerprinting: comparative diversity assessment against bacterial isolates and PCR-DNA clone libraries. Environ Microbiol 6, 95–110. [Google Scholar]
  17. Kanakaraj, R., Harris, D. L., Songer, J. G. & Bosworth, B.(1998). Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed. Vet Microbiol 63, 29–38.[CrossRef] [Google Scholar]
  18. Kikuchi, M., Ohnishi, K. & Harayama, S.(1999). Novel family shuffling methods for the in vitro evolution of enzymes. Gene 236, 159–167.[CrossRef] [Google Scholar]
  19. Kita, A., Kita, S., Fujisawa, I., Inaka, K., Ishida, T., Horiike, K., Nozaki, M. & Miki, K.(1999). An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Pseudomonas putida mt-2. Structure Fold Des 7, 25–34.[CrossRef] [Google Scholar]
  20. Kitayama, A., Achioku, T., Yanagawa, T. & 7 other authors(1996). Cloning and characterization of extradiol aromatic ring-cleavage dioxygenases of Pseudomonas aeruginosa JI104. J Ferment Bioeng 82, 217–223.[CrossRef] [Google Scholar]
  21. Kraulis, P. J.(1991). Molscript: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24, 946–950.[CrossRef] [Google Scholar]
  22. Kraut, D. A., Carroll, K. S. & Herschlag, D.(2003). Challenges in enzyme mechanism and energetics. Annu Rev Biochem 72, 517–571.[CrossRef] [Google Scholar]
  23. Kukor, J. J. & Olsen, R. H.(1996). Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments. Appl Environ Microbiol 62, 1728–1740. [Google Scholar]
  24. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef] [Google Scholar]
  25. Lamanda, A., Zahn, A., Roder, D. & Langen, H.(2004). Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-to-background ratio and improved baseline resolution. Proteomics 4, 599–608.[CrossRef] [Google Scholar]
  26. McKay, D. B., Prucha, M., Reineke, W., Timmis, K. N. & Pieper, D. H.(2003). Substrate specificity and expression of three 2,3-dihydroxybiphenyl 1,2-dioxygenases from Rhodococcus globerulus strain P6. J Bacteriol 185, 2944–2951.[CrossRef] [Google Scholar]
  27. Nakai, C., Kagamiyama, H., Nozaki, M., Nakazawa, T., Inouye, S., Ebina, Y. & Nakazawa, A.(1983). Complete nucleotide sequence of the metapyrocatechase gene on the TOL plasmid of Pseudomonas putida mt-2. J Biol Chem 258, 2923–2928. [Google Scholar]
  28. Nakajima, H., Ishida, T., Tanaka, H. & Horiike, K.(2002). Accurate measurement of near-micromolar oxygen concentrations in aqueous solutions based on enzymatic extradiol cleavage of 4-chlorocatechol: applications to improved low-oxygen experimental systems and quantitative assessment of back diffusion of oxygen from the atmosphere. J Biochem (Tokyo) 131, 523–531.[CrossRef] [Google Scholar]
  29. Nicholls, A., Sharp, K. A. & Honig, B.(1991). Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 282–296. [Google Scholar]
  30. Okuta, A., Ohnishi, K. & Harayama, S.(1998). PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes. Gene 212, 221–228.[CrossRef] [Google Scholar]
  31. Parales, R. E., Parales, J. V. & Gibson, D. T.(1999). Aspartate 205 in the catalytic domain of naphthalene dioxygenase is essential for activity. J Bacteriol 181, 1831–1837. [Google Scholar]
  32. Parales, R. E., Resnick, S. M., Yu, C. L., Boyd, D. R., Sharma, N. D. & Gibson, D. T.(2000). Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit. J Bacteriol 182, 5495–5504.[CrossRef] [Google Scholar]
  33. Pollmann, K., Wray, V., Hecht, H. J. & Pieper, D. H.(2003). Rational engineering of the regioselectivity of TecA tetrachlorobenzene dioxygenase for the transformation of chlorinated toluenes. Microbiology 149, 903–913.[CrossRef] [Google Scholar]
  34. Que, J. L. & Ho, R. Y. N.(1996). Dioxygen activation by enzymes with mononuclear non-heme iron active sites. Chem Rev 96, 2607–2624.[CrossRef] [Google Scholar]
  35. Rabilloud, T., Strub, J. M., Luche, S., van Dorsselaer, A. & Lunardi, J.(2001). A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. Proteomics 1, 699–704.[CrossRef] [Google Scholar]
  36. Ramos, J. L., Mermod, N. & Timmis, K. N.(1987). Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas. Mol Microbiol 1, 293–300.[CrossRef] [Google Scholar]
  37. Reineke, W. & Knackmuss, H.-J.(1988). Microbial degradation of haloaromatics. Annu Rev Microbiol 42, 263–287.[CrossRef] [Google Scholar]
  38. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  39. Shimotohno, A., Oue, S., Yano, T., Kuramitsu, S. & Kagamiyama, H.(2001). Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design. J Biochem 129, 943–948.[CrossRef] [Google Scholar]
  40. Shingler, V., Powlowski, J. & Marklund, U.(1992). Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. J Bacteriol 174, 711–724. [Google Scholar]
  41. Shu, L., Chiou, Y. M., Orville, A. M., Miller, M. A., Lipscomb, J. D. & Que, L., Jr(1995). X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry 34, 6649–6659.[CrossRef] [Google Scholar]
  42. Smith, M. R.(1990). The biodegradation of aromatic hydrocarbons by bacteria. Biodegradation 1, 191–206.[CrossRef] [Google Scholar]
  43. Spiller, B., Gershenson, A., Arnold, F. H. & Stevens, R. C.(1999). A structural view of evolutionary divergence. Proc Natl Acad Sci U S A 96, 12305–12310.[CrossRef] [Google Scholar]
  44. Vaillancourt, F. H., Labbe, G., Drouin, N. M., Fortin, P. D. & Eltis, L. D.(2002). The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2- dioxygenase by catecholic substrates. J Biol Chem 277, 2019–2027.[CrossRef] [Google Scholar]
  45. Wasserfallen, A., Rekik, M. & Harayama, S.(1991). A Pseudomonas putida strain able to degrade m-toluate in the presence of 3-chlorocatechol. Bio/Technology 9, 296–298.[CrossRef] [Google Scholar]
  46. Williams, P. A., Assinder, S. J. & Shaw, L. E.(1990). Construction of hybrid xylE genes between the two duplicate homologous genes from TOL plasmid pWW53: comparison of the kinetic properties of the gene products. J Gen Microbiol 136, 1583–1589.[CrossRef] [Google Scholar]
  47. Yang, H., Carr, P. D., McLoughlin, S. Y. & 7 other authors(2003). Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution. Protein Eng 16, 135–145.[CrossRef] [Google Scholar]
  48. Zielinski, M., Kahl, S., Hecht, H. J. & Hofer, B.(2003). Pinpointing biphenyl dioxygenase residues that are crucial for substrate interaction. J Bacteriol 185, 6976–6980.[CrossRef] [Google Scholar]

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