Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen
AUTOR(ES)
Ge, Yong
FONTE
American Society for Microbiology
RESUMO
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 Fe2S2 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, kcat = 3.9 ± 0.2 s−1, and KmO2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where KmO2 represents the Km for O2 and KmA represents the Km 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 O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate. However, the apparent Km of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135208Documentos Relacionados
- Biotransformation of substituted benzoates to the corresponding cis-diols by an engineered strain of Pseudomonas oleovorans producing the TOL plasmid-specified enzyme toluate-1,2-dioxygenase.
- Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates.
- Characterization of Hybrid Toluate and Benzoate Dioxygenases
- Dehalogenation, Denitration, Dehydroxylation, and Angular Attack on Substituted Biphenyls and Related Compounds by a Biphenyl Dioxygenase
- Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls