Purification of 3,5-dichlorocatechol 1,2-dioxygenase, a nonheme iron dioxygenase and a key enzyme in the biodegradation of a herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90

Manzoor A. Bhat, Tetsuo Ishida, Kihachiro Horiike, Chelakara S. Vaidyanathan, Mitsuhiro Nozaki

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Abstract

An enzyme which cleaves the benzene ring of 3,5-dichiorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 μM-1 s-1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the Mr value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s-1 and 4.4 and 652 μM, respectively, at pH 8 and 25°C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.

Original languageEnglish (US)
Pages (from-to)738-746
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume300
Issue number2
DOIs
StatePublished - Feb 1 1993
Externally publishedYes

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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