Overexpression of Pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered Escherichia coli.
The cloned xylE gene encoding catechol 2,3-dioxygenase (metapyrocatechase) from TOL plasmid in Pseudomonas putida mt-2 has been expressed in Escherichia coli W3110 to a level of approximately 15% of the total soluble protein. Of the total iron in the crude extract, 45% was on the enzyme. The crystallized enzyme from E. coli had higher iron content (3.7 mol/mol enzyme) and specific activity (536 U/mg) than the enzyme from P. putida mt-2. However, no differences were observed in physicochemical, protein-chemical, and kinetic properties between the two enzymes. The enzyme was a homotetramer, and no changes were observed in the values of M(r) (136,000 +/- 5,000) and Stokes radius (4.26 nm) in the concentration range from 0.36 nM to 2.8 microM, indicating that the native enzyme neither dissociated into subunits nor polymerized in this range. The catalytic center activity and the Km values for catechol and dioxygen were 278 s-1, 1.87 and 7.45 microM, respectively, at pH 7.5 and 25 degrees C. The enzyme showed a broad substrate specificity. Among substrates, 4-methylcatechol and 4-chlorocatechol showed specificity constants (approximately 200 microM-1.s-1) higher than that for catechol. Acetone and phenol derivatives competitively inhibited the activity against catechol. The relationship between specific activity and iron content was not linear, suggesting some conformational changes in the partially iron-depleted enzyme.
Kobayashi
T
T
Department of Biochemistry, Shiga University of Medical Science.
Ishida
T
T
Horiike
K
K
Takahara
Y
Y
Numao
N
N
Nakazawa
A
A
Nakazawa
T
T
Nozaki
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D050557:Catechol 2,3-Dioxygenase; D003001:Cloning, Molecular; D049308:Dioxygenases; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005818:Genetic Engineering; D007501:Iron / Q000378:metabolism; D008970:Molecular Weight; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D016958:Pseudomonas putida; D011994:Recombinant Proteins / Q000096:biosynthesis
D000596:Amino Acids; D011994:Recombinant Proteins; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase