Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans Strain CA10

Hideaki Nojiri, Hiroko Taira, Kenichi Iwata, Kenichi Morii, Jeong Won Nam, Takako Yoshida, Hiroshi Habe, Shugo Nakamura, Kentaro Shimizu, Hisakazu Yamane, Toshio Omori

Research output: Contribution to journalArticle

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Abstract

2-Hydroxy-6-oxo-6-(2′-aminophenyl)-hexa-2,4-dienoic acid [6-(2′-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6- oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 μM and kcat of 2.14 (s-1) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25°C). The effect of the presence of an amino group or hydroxyl group at the 2′-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2′-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2′- hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2′-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.

Original languageEnglish
Pages (from-to)36-45
Number of pages10
JournalBioscience, Biotechnology and Biochemistry
Volume67
Issue number1
Publication statusPublished - 2003 Jan
Externally publishedYes

Fingerprint

Pseudomonas resinovorans
carbazoles
Hydrolases
hydrolases
Purification
Enzymes
Acids
acids
enzymes
Degradation
sodium phosphate
degradation
Aromatic compounds
Enzyme activity
Molecular orbitals
Hydroxyl Radical
Escherichia coli
aromatic compounds
Phosphates
Substitution reactions

Keywords

  • Carbazole
  • Dibenzofuran
  • Meta-cleavage compound hydrolase
  • Pseudomonas resinovorans strain CA10

ASJC Scopus subject areas

  • Food Science
  • Applied Microbiology and Biotechnology
  • Chemistry (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Bioengineering

Cite this

Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans Strain CA10. / Nojiri, Hideaki; Taira, Hiroko; Iwata, Kenichi; Morii, Kenichi; Nam, Jeong Won; Yoshida, Takako; Habe, Hiroshi; Nakamura, Shugo; Shimizu, Kentaro; Yamane, Hisakazu; Omori, Toshio.

In: Bioscience, Biotechnology and Biochemistry, Vol. 67, No. 1, 01.2003, p. 36-45.

Research output: Contribution to journalArticle

Nojiri, H, Taira, H, Iwata, K, Morii, K, Nam, JW, Yoshida, T, Habe, H, Nakamura, S, Shimizu, K, Yamane, H & Omori, T 2003, 'Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans Strain CA10', Bioscience, Biotechnology and Biochemistry, vol. 67, no. 1, pp. 36-45.
Nojiri, Hideaki ; Taira, Hiroko ; Iwata, Kenichi ; Morii, Kenichi ; Nam, Jeong Won ; Yoshida, Takako ; Habe, Hiroshi ; Nakamura, Shugo ; Shimizu, Kentaro ; Yamane, Hisakazu ; Omori, Toshio. / Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans Strain CA10. In: Bioscience, Biotechnology and Biochemistry. 2003 ; Vol. 67, No. 1. pp. 36-45.
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abstract = "2-Hydroxy-6-oxo-6-(2′-aminophenyl)-hexa-2,4-dienoic acid [6-(2′-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6- oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 μM and kcat of 2.14 (s-1) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25°C). The effect of the presence of an amino group or hydroxyl group at the 2′-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2′-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2′- hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2′-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.",
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AU - Nam, Jeong Won

AU - Yoshida, Takako

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