1887

Abstract

Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase α subunit (BphA1) containing segments from sp. strain LB400 and P6, and of a hydroxylase β subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substrate-binding site. Exchanges of neither the β subunit nor the N- and C-terminal regions of the α subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the α subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in or other host organisms.

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2002-08-01
2019-12-13
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References

  1. Asturias, J. A., Moore, E., Yakimov, M. M., Klatte, S. & Timmis, K. N. ( 1994; ). Reclassification of the polychlorinated biphenyl-degraders Acinetobacter sp. strain P6 and Corynebacterium sp. strain MB1 as Rhodococcus globerulus. Syst Appl Microbiol 17, 226-231.[CrossRef]
    [Google Scholar]
  2. Asturias, J. A., Diaz, E. & Timmis, K. N. ( 1995; ). The evolutionary relationship of biphenyl dioxygenase from Gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from Gram-negative bacteria. Gene 156, 11-18.[CrossRef]
    [Google Scholar]
  3. Barriault, D., Simard, C., Chatel, H. & Sylvestre, M. ( 2001; ). Characterization of hybrid biphenyl dioxygenases obtained by recombining Burkholderia sp. strain LB400 bphA with the homologous gene of Comamonas testosteroni B-356. Can J Microbiol 47, 1025-1032.[CrossRef]
    [Google Scholar]
  4. 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]
  5. Bopp, L. H. ( 1986; ). Degradation of highly chlorinated PCBs by Pseudomonas strain LB400. J Ind Microbiol 1, 23-29.[CrossRef]
    [Google Scholar]
  6. Boyd, D. R. & Sheldrake, G. N. ( 1998; ). The dioxygenase-catalyzed formation of vicinal cis-diols. Nat Prod Rep 15, 309-325.[CrossRef]
    [Google Scholar]
  7. Butler, C. S. & Mason, J. R. ( 1997; ). Structure-function analysis of the bacterial aromatic ring-hydroxylating dioxygenases. Adv Microb Physiol 38, 47-84.
    [Google Scholar]
  8. 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 Biol 296, 701–712.[CrossRef]
    [Google Scholar]
  9. Chebrou, H., Hurtubise, Y., Barriault, D. & Sylvestre, M. ( 1999; ). Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it. J Bacteriol 181, 4805-4811.
    [Google Scholar]
  10. Erickson, B. D. & Mondello, F. ( 1992; ). Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol 174, 2903-2912.
    [Google Scholar]
  11. Erickson, B. D. & Mondello, F. J. ( 1993; ). Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene. Appl Environ Microbiol 59, 3858-3862.
    [Google Scholar]
  12. Franklin, F. C. H., Bagdasarian, M., Bagdasarian, M. M. & Timmis, K. N. ( 1983; ). Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci USA 78, 7458-7462.
    [Google Scholar]
  13. Furukawa, K., Matsumura, F. & Tonomura, K. ( 1978; ). Alcaligenes and Acinetobacter strains capable of degrading polychlorinated biphenyls. Agric Biol Chem 42, 543-548.[CrossRef]
    [Google Scholar]
  14. Furukawa, K., Hirose, J., Hayashida, S. & Nakamura, K. ( 1994; ). Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase. J Bacteriol 176, 2121-2123.
    [Google Scholar]
  15. Gibson, D. T. & Parales, R. E. ( 2000; ). Aromatic hydrocarbon dioxygenases in environmental biotechnology. Curr Opin Biotechnol 11, 236-243.[CrossRef]
    [Google Scholar]
  16. Grant, S. G. N., Jesse, J., Bloom, F. R. & Hanahan, D. ( 1990; ). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87, 4645-4649.[CrossRef]
    [Google Scholar]
  17. Haddock, J. D., Horton, J. R. & Gibson, D. T. ( 1995a; ). Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. J Bacteriol 177, 5834-5839.
    [Google Scholar]
  18. Haddock, J. D., Horton, J. R. & Gibson, D. T. ( 1995b; ). Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. J Bacteriol 177, 20-26.
    [Google Scholar]
  19. Hanahan, D. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[CrossRef]
    [Google Scholar]
  20. Higuchi, R., Krummel, B. & Saiki, R. K. ( 1988; ). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16, 7351-7367.[CrossRef]
    [Google Scholar]
  21. Hirose, J., Suyama, A., Hayashida, S. & Furukawa, K. ( 1994; ). Construction of hybrid biphenyl (bph) and toluene (tod) genes for functional analysis of aromatic ring dioxygenases. Gene 138, 27-33.[CrossRef]
    [Google Scholar]
  22. Hofer, B., Eltis, L. D., Dowling, D. N. & Timmis, K. N. ( 1993; ). Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl (PCB) degradation. Gene 130, 47-55.[CrossRef]
    [Google Scholar]
  23. Hofer, B., Backhaus, S. & Timmis, K. N. ( 1994; ). The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes. Gene 144, 9-16.[CrossRef]
    [Google Scholar]
  24. Hudlicky, T., Gonzalez, D. & Gibson, D. T. ( 1999; ). Enzymatic hydroxylation of aromatics in enantioselective synthesis: expanding asymmetric methodology. Aldrichim Acta 32, 35-62.
    [Google Scholar]
  25. Hurtubise, Y., Barriault, D. & Sylvestre, M. ( 1998; ). Involvement of the terminal oxygenase beta subunit in the biphenyl dioxygenase reactivity pattern toward chlorobiphenyls. J Bacteriol 180, 5828-5835.
    [Google Scholar]
  26. Jiang, H., Parales, R. E., Lynch, N. A. & Gibson, D. T. ( 1996; ). Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites. J Bacteriol 178, 3133-3139.
    [Google Scholar]
  27. 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. Structure 6, 571-586.[CrossRef]
    [Google Scholar]
  28. Kimura, N., Nishi, A., Goto, M. & Furukawa, K. ( 1997; ). Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally. J Bacteriol 179, 3936-3943.
    [Google Scholar]
  29. Kumamaru, T., Suenaga, H., Mitsuoka, M., Watanabe, T. & Furukawa, K. ( 1998; ). Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase. Nat Biotechnol 16, 663-666.[CrossRef]
    [Google Scholar]
  30. McKay, D. B., Seeger, M., Zielinski, M., Hofer, B. & Timmis, K. N. ( 1997; ). Heterologous expression of biphenyl dioxygenase-encoding genes from a Gram-positive broad-spectrum PCB degrader and characterization of chlorobiphenyl oxidation by the gene products. J Bacteriol 179, 1924-1930.
    [Google Scholar]
  31. Mondello, F. J., Turcich, M. P., Lobos, J. H. & Erickson, B. D. ( 1997; ). Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation. Appl Environ Microbiol 63, 3096-3103.
    [Google Scholar]
  32. Parales, R. E., Lee, K., Resnick, S. M., Jiang, H., Lessner, D. J. & Gibson, D. T. ( 2000; ). Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. J Bacteriol 182, 1641-1649.[CrossRef]
    [Google Scholar]
  33. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. ( 1988; ). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491.[CrossRef]
    [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.
  35. Seeger, M., Timmis, K. N. & Hofer, B. ( 1995a; ). Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400. Appl Environ Microbiol 61, 2654-2658.
    [Google Scholar]
  36. Seeger, M., Timmis, K. N. & Hofer, B. ( 1995b; ). Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp. LB400. FEMS Microbiol Lett 133, 259-264.[CrossRef]
    [Google Scholar]
  37. Seeger, M., Zielinski, M., Timmis, K. N. & Hofer, B. ( 1999; ). Regiospecificity of dioxygenation of di- to pentachlorobiphenyls and their degradation to chlorobenzoates by the bph-encoded catabolic pathway of Burkholderia sp. strain LB400. Appl Environ Microbiol 65, 3614-3621.
    [Google Scholar]
  38. Studier, F. W. ( 1991; ). Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219, 37-44.[CrossRef]
    [Google Scholar]
  39. Tabor, S. & Richardson, C. C. ( 1985; ). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82, 1074-1078.[CrossRef]
    [Google Scholar]
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