Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning of p-Hydroxybenzoate Degradation Genes and the Overexpression of Protocatechuate 4,5-Dioxygenase from Pseudomonas sp. K82

  • Published : 2006.12.30
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Abstract

Pseudomonas sp. K82 cultured in p-hydroxybenzoate induces protocatechuate 4,5-dioxygenase (PCD 4,5) for p-hydroxybenzoate degradation. In this study, a 6.0-kbp EcoR1 fragment containing p-hydroxybenzoate degradation genes was cloned from the genome of Pseudomonas sp. K82. Sequence analysis identified four genes, namely, pcaD, pcaA, pcaB, and pcaC genes known to be involved in p-hydroxybenzoate degradation. Two putative 4-hydroxyphenylpyruvate dioxygenases and one putative oxidoreductase were closely located by the p-hydroxybenzoate degradation genes. The gene arrangement and sequences of these p-hydroxybenzoate degradation genes were similar to those of Comamonas testosteroni and Pseudomonas ochraceae. PcaAB (PCD4,5) was overexpressed in the expression vector pGEX-4T-3, purified using a GST column, and confirmed to have protocatechuate 4,5-dioxygenase activity. The N-terminal amino acid sequences of overexpressed PCD4,5 were identical with those of purified PCD4,5 from Pseudomonas sp. K82.

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References

  1. Arciero, D. M., A. M. Orville, and J. D. Lipscomb. 1990. Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni. Methods Enzymol. 188: 89-95 https://doi.org/10.1016/0076-6879(90)88017-5
  2. Cha, C. J. 2006. Catechol 1,2-dioxygenase from Rhodococcus rhodochrous N75 capable of metabolizing alkyl-substituted catechols. J. Microbiol. Biotechnol. 16: 778-785
  3. Chen, Y. P. and C. R. Lovell. 1994. Purification and properties of a homodimeric protocatechuate 4,5-dioxygenase from Rhizobium leguminosarum. Arch. Microbiol. 161: 191-195 https://doi.org/10.1007/BF00276482
  4. Harayama, S. and M. Kok. 1992. Functional and evolutionary relationships among diverse oxygenases. Annu. Rev. Microbiol. 46: 565-601 https://doi.org/10.1146/annurev.mi.46.100192.003025
  5. Kim, S. I., J. Y. Kim, S.-H. Yun, J. H. Kim, S.-H. Leem, and C.-H. Lee. 2004. Proteome analysis of Pseudomonas sp. K82 biodegradation pathways. Proteomics 4: 3610-3621 https://doi.org/10.1002/pmic.200400977
  6. Kim, S.-J., D. Y. Jun, C. H. Yang, and Y. H. Kim. 2006. Cloning and expression of hpaA gene of Korean strain Helicobacter pylori K51 in oral vaccine delivery vehicle Lactococcus lactis subsp. lactis MG1363. J. Microbiol. Biotechnol. 16: 318-324 https://doi.org/10.1159/000098281
  7. Mampel, J., M. A. Providenti, and A. M. Cook. 2005. Protocatechuate 4,5-dioxygenase from Comamonas testosterone T-2: Biochemical and molecular properties of a new subgroup within class II of extradiol dioxygenases. Arch. Microbiol. 183: 130-139 https://doi.org/10.1007/s00203-004-0755-4
  8. Maruyama, K., T. Shibayama, A. Ichikawa, Y. Sakou, S. Yamada, and H. Sugisaki. 2004. Cloning and characterization of the genes encoding enzymes for the protocatechuate meta-degradation pathway of Pseudomonas ochraceae NGJ1. Biosci. Biotechnol. Biochem. 68: 1434-1441 https://doi.org/10.1271/bbb.68.1434
  9. Moran, G. R. 2005. 4-Hydroxyphenylpyruvate dioxygenase. Arch. Biochem. Biophys. 433: 117-128 https://doi.org/10.1016/j.abb.2004.08.015
  10. Noda, Y., S. Nichikawa, K. Ahiozuka, H. Kandokura, H. Hakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. 1990. Molecular cloning of protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis. J Bacteriol. 172: 2704-2709 https://doi.org/10.1128/jb.172.5.2704-2709.1990
  11. Ono, K., M. Nozaki, and O. Hayaishi. 1970. Purification and some properties of protocatechuate4,5-dioxygenase. Biochim. Biophys. Acta 22: 224-238
  12. Providenti, M. A., J. Mampel, S. MacSween, A. M. Cook, and R. C. Wyndham. 2001. Comamonas testosteroni BR6020 possesses a single genetic locus for extradiol cleavage of protocatechuate. Microbiology 147: 2157-2167 https://doi.org/10.1099/00221287-147-8-2157
  13. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  14. Spence, E. L., M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg. 1996. Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): Sequence analysis and biochemical properties of a third family of extradiol dioxygenases. J. Bacteriol. 178: 5249- 5256 https://doi.org/10.1128/jb.178.17.5249-5256.1996
  15. Sugimoto, K., T. Senda, H. Aoshima, E. Masai, M. Fukuda, and Y. Mitsui. 1999. Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions. Structure 7: 953-965 https://doi.org/10.1016/S0969-2126(99)80122-1
  16. Wattiau, P., L. Bastianes, R. Herwijnen, L. Daal, J. R. Parsons, M.-E. Renard, D. Springael, and G. R. Cornelis. 2001. Fluorene degradation by Sphingomonas sp. LB126 proceeds through protocatechuate acid: A genetic analysis. Res. Microbiol. 152: 861-872 https://doi.org/10.1016/S0923-2508(01)01269-4
  17. Yun, S.-H., C.-Y. Yun, and S. I. Kim. 2004. Characterization of protocatechuate 4,5-dioxygenase induced from phydroxybenzoate- cultured Pseudomonas sp. K82. J. Microbiol. 42: 152-155