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Journal of Bacteriology, September 2003, p. 5333-5341, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5333-5341.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Hybrid Toluate and Benzoate Dioxygenases

Yong Ge and Lindsay D. Eltis^*

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received 21 February 2003/ Accepted 24 June 2003

Toluate dioxygenase of Pseudomonas putida mt-2 (TADO_mt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADO_ADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the subunit of one enzyme and the ß subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADO_ADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADO_mt2 (3-methylbenzoate > benzoate 3-chlorobenzoate > 4-methylbenzoate 4-chlorobenzoate >> 2-methylbenzoate 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the _Bß_T hybrid oxygenase for these benzoates corresponded to that of BADO_ADP1, the parent from which the subunit originated. In contrast, the apparent substrate specificity of the _Tß_B hybrid oxygenase differed slightly from that of TADO_mt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the _Tß_B hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADO_mt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O₂ utilization in the hybrid than in the parent. Overall, these results support the notion that the subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the ß subunit contributes significantly to the enzyme's function.

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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.

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Journal of Bacteriology, September 2003, p. 5333-5341, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5333-5341.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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