%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