Reactivity of toluate dioxygenase with substituted benzoates and dioxygen.
Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe(2)S(2) centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 +/- 1 microM, k(cat) = 3.9 +/- 0.2 s(-1), and K(m)O(2) = 16 +/- 2 microM (100 mM sodium phosphate, pH 7.0; 25 degrees C), where K(m)O(2) represents the K(m) for O(2) and KmA represents the K(m) for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate approximately 3-chlorobenzoate > p-toluate approximately 4-chlorobenzoate >> o-toluate approximately 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O(2) utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O(2) utilization, with >10 times more O(2) being consumed than benzoate. However, the apparent K(m) of TADO for these benzoates was >100 microM, indicating that they do not effectively inhibit the turnover of good substrates.
Ge
Yong
Y
Department of Microbiology and Immunology, University of British Columbia, Vancouver BC V6T 1Z3, Canada.
Vaillancourt
Frédéric H
FH
Agar
Nathalie Y R
NY
Eltis
Lindsay D
LD
D001565:Benzoates / Q000494:pharmacology*; D002723:Chlorobenzoates / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D010100:Oxygen / Q000494:pharmacology*; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D001565:Benzoates; D002723:Chlorobenzoates; D011994:Recombinant Proteins; C036427:3-chlorobenzoic acid; C009503:3-toluic acid; C060415:2-chlorobenzoic acid; D010105:Oxygenases; C023172:toluene dioxygenase; C040768:4-chlorobenzoic acid; D010100:Oxygen