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Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

2002; American Society for Microbiology; Volume: 184; Issue: 15 Linguagem: Inglês

10.1128/jb.184.15.4096-4103.2002

1098-5530

Yong Ge, Frédéric H. Vaillancourt, Nathalie Y.R. Agar, Lindsay D. Eltis,

Pesticide and Herbicide Environmental Studies

ABSTRACT 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 μM , k cat = 3.9 ± 0.2 s −1 , and K m O 2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°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 ≃ 3-chlorobenzoate > p -toluate ≃ 4-chlorobenzoate ≫ o -toluate ≃ 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 μM, indicating that they do not effectively inhibit the turnover of good substrates.