Archives of Microbiology

, Volume 183, Issue 1, pp 1–8

A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli

  • Jian-Feng Wu
  • Cui-Wei Sun
  • Cheng-Ying Jiang
  • Zhi-Pei Liu
  • Shuang-Jiang Liu
Original Paper
  • 331 Downloads

Abstract

Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of α- and β-subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (Km=0.77 μM) than for 2-aminophenol (Km=0.89 μM) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the α- and β-subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the β- and α-subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.

Keywords

Comamonas 2-Aminophenol 1 6-Dioxygenase Chloronitrobenzene degradation 

References

  1. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  2. Davis JK, He Z, Somerville CC, Spain JC (1999) Genetic and biochemical comparison of 2-aminophenol 1,6-dioxygenase of Pseudomonas pseudoalcaligenes JS45 to meta-cleaveage dioxygenases: divergent evolution of 2-aminophenol meta-cleavage pathway. Arch Microbiol 172:330–339CrossRefPubMedGoogle Scholar
  3. Davison AD, Karuso P, Jardine DR, Veal DA (1996) Halopicolinic acids, novel products arising through the degradation of chloro-and bromobiphenyl by Sphingomonas paucimobilis BPSI-3. Can J Microbiol 42:66–71PubMedGoogle Scholar
  4. European Economic Community (1982) Communication from the commission to the council on dangerous substances which might be included in list I of council directive 76/464/EEC. European Economic Community, BrusselsGoogle Scholar
  5. Häggblom MM (1992) Microbial breakdown of halogenated aromatic pesticides and related compounds. FEMS Microbiol Rev 103:29–72CrossRefGoogle Scholar
  6. Iwagami SG, Yang K, Davies J (2000) Characterization of the protocatechuic acid catabolic gene cluster from Streptomyces sp. strain 2065. Appl Environ Microbiol 66:1499–1508CrossRefPubMedGoogle Scholar
  7. Johnson GR, Spain JC (2003) Evolution of catabolic pathways for synthetic compounds: bacterial pathways for degradation of 2,4-dinitrotolunene and nitrobenzene. Appl Microbiol Biotechnol 62:110–123CrossRefPubMedGoogle Scholar
  8. Katsivela E, Wray V, Pieper DH, Wittich R-M (1999) Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1. Appl Environ Microbiol 65:1405–1412PubMedGoogle Scholar
  9. Konopka A (1993) Isolation and characterization of subsurface bacterium that degrades aniline and methylanilines. FEMS Microbiol Lett 111:93–100CrossRefGoogle Scholar
  10. Lendenmann U, Spain JC (1996) 2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45. J Bacteriol 178:6227–6232PubMedGoogle Scholar
  11. Linch AL (1974) Biological monitoring for industrial exposure to cyanogenic aromatic nitro and amino compounds. Am Ind Hyg Assoc J 62:784–790Google Scholar
  12. Livingston AG, Brookes PR (1994) Biological detoxification of a 3-chloronitrobenzene manufacture wastwater in an extractive membrane bioreactor. Water Res 28:1347–1354CrossRefGoogle Scholar
  13. Park H-S, Kim H-S (2000) Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12. J Bacteriol 182:573–580CrossRefPubMedGoogle Scholar
  14. Park H-S, Lim S-J, Chang YK, Livingston AG, Kim H-S (1999) Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and a Rhodococcus sp. Appl Envrion Microbiol 65:1083–1091Google Scholar
  15. Sala-Trepat JM, Evans WC (1971) The meta-cleavage of catechol by Azobacter species. Eur J Chem 20:400–413Google Scholar
  16. Sambrook J, Russell DW (2001) In: Molecular Cloning, 3rd edn, vol 1. CSHL Press, Cold Spring Harbor, New York, pp 4.11–4.24Google Scholar
  17. Shimizu M, Yasui T, Matsumoto N (1983) Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium-a series of chloro- or fluoro-nitrobenzene derivatives. Mutat Res 116:217–238PubMedGoogle Scholar
  18. Spence EL, Kawamukai M, Sanvoisin J, Braven H, Bugg TDH (1996) Catechol dioxygenases from Escherichia coli (MphB) ad Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases. J Bacteriol 178:5249–5256PubMedGoogle Scholar
  19. Sugimoto K, Senda T, Aoshima H, Masai E, Fukuda M, Mitsui Y (1999) Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions. Structure 7:953–965CrossRefPubMedGoogle Scholar
  20. Takenaka S, Murakami S, Shinke R, Hatakeyama K, Yukawa H, Aoki K (1997) Novel genes encoding 2-aminophenol 1,6-dioxygenase from Pseudomonas species AP-3 growing on 2-aminopheol and catalytic properties of the purified enzyme. J Biol Chem 272:14727–14732CrossRefPubMedGoogle Scholar
  21. Takenaka S, Murakami S, Kim Y-J, Aoki K (2000) Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3. Arch Microbiol 174:265–272CrossRefPubMedGoogle Scholar
  22. Weisburger EK, Russfield F, Homburger F, Weisburger JH, Boger E, Van Dongen CG, Chu KC (1978) Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity. Environ Pathol Toxicol 2:325–356Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Jian-Feng Wu
    • 1
  • Cui-Wei Sun
    • 1
  • Cheng-Ying Jiang
    • 1
  • Zhi-Pei Liu
    • 1
  • Shuang-Jiang Liu
    • 1
  1. 1.Institute of MicrobiologyChinese Academy of SciencesBeijingChina

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