1887

Abstract

The tetrachlorobenzene dioxygenase (TecA) of sp. PS12 carries out the first step in the aerobic biodegradation of chlorinated toluenes. Besides dioxygenation of the aromatic ring of 4-chloro-, 2,4-, 2,5- and 3,4-dichlorotoluene as the main reaction, it also catalyses mono-oxygenation of the methyl groups of 2,3-, 2,6-, 3,5-di- and 2,4,5-trichlorotoluene as the main reactions, channelling these compounds into dead-end pathways. Based on the crystal structure of the homologous naphthalene dioxygenase (NDO) and alignment of the -subunits of NDO and TecA, the substrate pocket of TecA was modelled. Recently, for NDO and the homologous 2-nitrotoluene dioxygenase (2NTDO), two amino acids (Phe of NDO and Asn of 2NTDO) were identified which control the regioselectivity of these enzymes. The corresponding amino acids at Phe and Leu of TecA were substituted to change the regioselectivity and to expand the product spectrum. Position 366 was shown to control regioselectivity of the enzyme, although mutations resulted in decreased or lost activity. Amino acid substitutions at Leu had little or no effect on the regioselectivity of TecA, but had significant effects on the product formation rate. Substitutions at both positions changed the site of oxidation of 2,4,5-trichlorotoluene slightly. As new products, 3,4,6-trichloro-1-methyl-1,2-dihydroxy-1,2-dihydrocyclohexan-3,5-diene, 4,6-dichloro-3-methylcatechol, 3,6-dichloro-4-methylcatechol and 3,4-dichloro-6-methylcatechol were identified.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26054-0
2003-04-01
2020-04-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/4/mic149903.html?itemId=/content/journal/micro/10.1099/mic.0.26054-0&mimeType=html&fmt=ahah

References

  1. Beil S., Happe B., Timmis K. N., Pieper D. H.. 1997; Genetic and biochemical characterization of the broad-spectrum chlorobenzene dioxygenase from Burkholderia sp. strain PS12: dechlorination of 1,2,4,5-tetrachlorobenzene. EurJ Biochem247:190–199
    [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 Bacteriol180:5520–5528
    [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 Biochem72:248–254
    [Google Scholar]
  4. Brühlmann F., Chen W.. 1999; Tuning biphenyl dioxygenase for extended substrate specificity. Biotechnol Bioeng63:544–551
    [Google Scholar]
  5. Carredano E., Karlsson A., Kauppi B.. 7 other authors 2000; Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding. J Mol Biol296:701–712
    [Google Scholar]
  6. Ensley B. D., Gibson D. T.. 1983; Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. J Bacteriol155:505–511
    [Google Scholar]
  7. Ensley B. D., Gibson D. T., Laborde A. L.. 1982; Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816. J Bacteriol149:948–954
    [Google Scholar]
  8. Erickson B. D., Mondello F. J.. 1992; Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol174:2903–2912
    [Google Scholar]
  9. Erickson B. D., Mondello F. J.. 1993; Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene. Appl Environ Microbiol59:3858–3862
    [Google Scholar]
  10. Gibson D. T., Parales R. E.. 2000; Aromatic hydrocarbon dioxygenases in environmental biotechnology. Curr Opin Biotechnol11:236–243
    [Google Scholar]
  11. Harayama S., Rekik M., Timmis K. N.. 1986; Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida . Mol Gen Genet202:226–234
    [Google Scholar]
  12. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R.. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene77:61–68
    [Google Scholar]
  13. Irie S., Doi S., Yorifuji T., Takagi M., Yano K.. 1987; Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida . J Bacteriol169:5174–5179
    [Google Scholar]
  14. Jones T., Zou J., Cowan S., Kjeldgaard M.. 1991; Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A47:110–119
    [Google Scholar]
  15. Kauppi B., Lee K., Carredano E., Parales R. E., Gibson D. T., Eklund H., Ramaswamy S.. 1998; Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase. Structure6:571–586
    [Google Scholar]
  16. Kirsch N. H., Stan H.-J.. 1994; Gas chromatographic-mass spectrometric determination of chlorinated cis -1,2-dihydrocyclohexadienes and chlorocatechols as their boronates. J Chromatogr A684:277–287
    [Google Scholar]
  17. Kraulis P. J.. 1991; molscript: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr24:946–950
    [Google Scholar]
  18. Kurkela S., Lehväslaiho H., Palva E. T., Teeri T. H.. 1988; Cloning, nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816. Gene73:355–362
    [Google Scholar]
  19. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685
    [Google Scholar]
  20. Laskowski R. A., MacArthur M. W., Moss D. S., Thornton J. M.. 1993; procheck: a program to check the stereochemical quality of protein structures. J Appl Crystallogr26:283–291
    [Google Scholar]
  21. Lee K., Gibson D. T.. 1996; Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. Appl Environ Microbiol62:3101–3106
    [Google Scholar]
  22. Lehning A., Fock U., Wittich R.-M., Timmis K. N., Pieper D. H.. 1997; Metabolism of chlorotoluenes by Burkholderia sp. PS12 and toluene dioxygenase of Pseudomonas putida F1: evidence for monooxygenation by toluene and chlorobenzene dioxygenases. Appl Environ Microbiol63:1974–1979
    [Google Scholar]
  23. Morris G. M., Goodsell D. S., Halliday R. S., Huey R., Hart W. E., Belew R. K., Olson A. J.. 1998; Automated docking using a lamarckian genetic algorithm and empirical binding free energy function. J Comput Chem19:1639–1662
    [Google Scholar]
  24. Neidle E. L., Hartnett C., Ornston N., Bairoch A., Rekik M., Harayama S.. 1991; Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases. J Bacteriol173:5385–5395
    [Google Scholar]
  25. Nicholls A., Sharp K. A., Honig B.. 1991; Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins11:282–296
    [Google Scholar]
  26. Parales J. V., Kumar A., Parales R. E., Gibson D. T.. 1996; Cloning and sequencing of the genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp. JS42. Gene181:57–61
    [Google Scholar]
  27. Parales R. E., Emig M. D., Lynch N. A., Gibson D. T.. 1998; Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems. J Bacteriol180:2337–2344
    [Google Scholar]
  28. Parales R. E., Lee K., Resnick S. M., Jiang H. Y., Lessner D. J., Gibson D. T.. 2000a; Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. J Bacteriol182:1641–1649
    [Google Scholar]
  29. Parales R. E., Resnick S. M., Yu C. L., Boyd D. R., Sharma N. D., Gibson D. T.. 2000b; Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis -dihydroxylation: control by phenylalanine 352 in the alpha subunit. J Bacteriol182:5495–5504
    [Google Scholar]
  30. Pollmann K., Beil S., Pieper D. H.. 2001; Transformation of chlorinated benzenes and toluenes by Ralstonia sp. strain PS12 tec A (tetrachlorobenzene dioxygenase) and tec B (chlorobenzene dihydrodiol dehydrogenase) gene products. Appl Environ Microbiol67:4057–4063
    [Google Scholar]
  31. Pollmann K., Kaschabek S., Wray V., Reineke W., Pieper D. H.. 2002; Metabolism of dichloromethylcatechols as central intermediates in the degradation of dichlorotoluenes by Ralstonia sp. strain PS12. J Bacteriol184:5261–5274
    [Google Scholar]
  32. Sakamoto T., Joern J. M., Arisawa A., Arnold F. H.. 2001; Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate. Appl Environ Microbiol67:3882–3887
    [Google Scholar]
  33. Sali A., Blundell T. L.. 1993; Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol234:779–815
    [Google Scholar]
  34. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Sander P., Wittich R.-M., Fortnagel P., Wilkes H., Francke W.. 1991; Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains. Appl Environ Microbiol57:1430–1440
    [Google Scholar]
  36. Shao Z., Arnold F. H.. 1996; Engineering new functions and altering existing functions. Curr Opin Struct Biol6:513–518
    [Google Scholar]
  37. Simon M. J., Osslund T. D., Saunders R.. 7 other authors 1993; Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4. Gene127:31–37
    [Google Scholar]
  38. Suenaga H., Mitsuoka M., Ura Y., Watanabe T., Furukawa K.. 2001; Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes. J Bacteriol183:5441–5444
    [Google Scholar]
  39. Tan H.-M., Cheong C.-M.. 1994; Substitution of the ISP α subunit of biphenyl dioxygenase from Pseudomonas results in a modification of the enzyme activity. Biochem Biophys Res Commun204:912–917
    [Google Scholar]
  40. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G.. 1997; The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res25:4876–4882
    [Google Scholar]
  41. Werlen C., Kohler H. P. E., van der Meer J. R.. 1996; The broad substrate chlorobenzene dioxygenase and cis -chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation. J Biol Chem271:4009–4016
    [Google Scholar]
  42. Zylstra G. J., Gibson D. T.. 1989; Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the tod C1C2BADE genes and their expression in Escherichia coli . J Biol Chem264:14940–14946
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26054-0
Loading
/content/journal/micro/10.1099/mic.0.26054-0
Loading

Data & Media loading...

Most cited this month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error