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Journal of Bacteriology, August 2002, p. 4096-4103, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4096-4103.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

Yong Ge,^1,2 Frédéric H. Vaillancourt,^1,2 Nathalie Y. R. Agar,^2, and Lindsay D. Eltis^1,2*

Department of Microbiology and Immunology, University of British Columbia, Vancouver BC V6T 1Z3,¹ Department of Biochemistry, Université Laval, Quebec City, Quebec G1K 7P4, Canada²

Received 22 February 2002/ Accepted 6 May 2002

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, k_cat = 3.9 ± 0.2 s^-1, and K_mO₂ = 16 ± 2 µM (100 mM sodium phosphate, pH 7.0; 25°C), where K_mO₂ represents the K_m for O₂ 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 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 K_m of TADO for these benzoates was >100 µM, indicating that they do not effectively inhibit the turnover of good substrates.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver BC V6T 1Z3, Canada. Phone: (604) 822-0042. Fax: (604) 822-6041. E-mail: leltis{at}interchange.ubc.ca.

Present address: Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H3G 1M8, Canada.

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Journal of Bacteriology, August 2002, p. 4096-4103, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4096-4103.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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