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.
An enzyme which cleaves the benzene ring of 3,5-dichlorocatechol 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 microM-1s-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 M(r) 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 microM, respectively, at pH 8 and 25 degrees 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.
Bhat
M A
MA
Department of Biochemistry, Shiga University of Medical Science, Japan.
Ishida
T
T
Horiike
K
K
Vaidyanathan
C S
CS
Nozaki
M
M
D015084:2,4-Dichlorophenoxyacetic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D016956:Burkholderia cepacia / Q000201:enzymology*; D002413:Cations, Divalent; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D049308:Dioxygenases; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity
D002413:Cations, Divalent; D046911:Macromolecular Substances; D015084:2,4-Dichlorophenoxyacetic Acid; D010105:Oxygenases; C079636:3,5-dichlorocatechol 1,2-dioxygenase; D049308:Dioxygenases