%0 Electronic Article %A Ge, Yong and Eltis, Lindsay D. %I American Society for Microbiology %D 2003 %D 2003 %G English %@ 0021-9193 %@ 1098-5530 %~ Katalog der Westsächsischen Hochschule Zwickau %T Characterization of Hybrid Toluate and Benzoate Dioxygenases %V 185 %J Journal of Bacteriology %V 185 %N 18 %P 5333-5341 %U http://dx.doi.org/10.1128/jb.185.18.5333-5341.2003 %X ABSTRACT 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 α subunit of one enzyme and the β 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 α B β T hybrid oxygenase for these benzoates corresponded to that of BADO ADP1 , the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the α T β 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 α T β 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 O 2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function. %Z https://www.katalog.fh-zwickau.de/Record/ai-49-aHR0cDovL2R4LmRvaS5vcmcvMTAuMTEyOC9qYi4xODUuMTguNTMzMy01MzQxLjIwMDM %U https://www.katalog.fh-zwickau.de/Record/ai-49-aHR0cDovL2R4LmRvaS5vcmcvMTAuMTEyOC9qYi4xODUuMTguNTMzMy01MzQxLjIwMDM