Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1.
Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2. Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 [Y. Kojima et al. (1967) J. Biol. Chem. 242, 3270-3278]. These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants. The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000. The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation. During the degradation, a single amino acid was released at each step. The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine. The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis. These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta [C. Nakai et al. (1979) Arch. Biochem. Biophys. 195, 12-22], although these two enzymes have very similar properties.
Nakai
C
C
Department of Biochemistry, Shiga University of Medical Science, Japan.
Nakazawa
T
T
Nozaki
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000917:Antibody Formation; D050548:Catechol 1,2-Dioxygenase; D049308:Dioxygenases; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification*; D011233:Precipitin Tests; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000596:Amino Acids; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase