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Characterization of Hybrid Toluate and Benzoate Dioxygenases
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| Zeitschriftentitel: | Journal of Bacteriology |
|---|---|
| Personen und Körperschaften: | , |
| In: | Journal of Bacteriology, 185, 2003, 18, S. 5333-5341 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
|
| Schlagwörter: |
| author_facet |
Ge, Yong Eltis, Lindsay D. Ge, Yong Eltis, Lindsay D. |
|---|---|
| author |
Ge, Yong Eltis, Lindsay D. |
| spellingShingle |
Ge, Yong Eltis, Lindsay D. Journal of Bacteriology Characterization of Hybrid Toluate and Benzoate Dioxygenases Molecular Biology Microbiology |
| author_sort |
ge, yong |
| spelling |
Ge, Yong Eltis, Lindsay D. 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.185.18.5333-5341.2003 <jats:title>ABSTRACT</jats:title> <jats:p> Toluate dioxygenase of <jats:italic>Pseudomonas putida</jats:italic> mt-2 (TADO <jats:sub>mt2</jats:sub> ) and benzoate dioxygenase of <jats:italic>Acinetobacter calcoaceticus</jats:italic> ADP1 (BADO <jats:sub>ADP1</jats:sub> ) 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 <jats:sub>ADP1</jats:sub> 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 <jats:sub>mt2</jats:sub> (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 <jats:sub>B</jats:sub> β <jats:sub>T</jats:sub> hybrid oxygenase for these benzoates corresponded to that of BADO <jats:sub>ADP1</jats:sub> , the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the α <jats:sub>T</jats:sub> β <jats:sub>B</jats:sub> hybrid oxygenase differed slightly from that of TADO <jats:sub>mt2</jats:sub> (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the α <jats:sub>T</jats:sub> β <jats:sub>B</jats:sub> hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADO <jats:sub>mt2</jats:sub> parent. Finally, the turnover of this <jats:italic>ortho</jats:italic> -substituted benzoate was much better coupled to O <jats:sub>2</jats:sub> 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. </jats:p> Characterization of Hybrid Toluate and Benzoate Dioxygenases Journal of Bacteriology |
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10.1128/jb.185.18.5333-5341.2003 |
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Online Free |
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Biologie |
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ElectronicArticle |
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American Society for Microbiology, 2003 |
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American Society for Microbiology, 2003 |
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0021-9193 1098-5530 |
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0021-9193 1098-5530 |
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ge2003characterizationofhybridtoluateandbenzoatedioxygenases |
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2003 |
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American Society for Microbiology |
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ai |
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ai |
| series |
Journal of Bacteriology |
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49 |
| title |
Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_unstemmed |
Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_full |
Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_fullStr |
Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_full_unstemmed |
Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_short |
Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_sort |
characterization of hybrid toluate and benzoate dioxygenases |
| topic |
Molecular Biology Microbiology |
| url |
http://dx.doi.org/10.1128/jb.185.18.5333-5341.2003 |
| publishDate |
2003 |
| physical |
5333-5341 |
| description |
<jats:title>ABSTRACT</jats:title>
<jats:p>
Toluate dioxygenase of
<jats:italic>Pseudomonas putida</jats:italic>
mt-2 (TADO
<jats:sub>mt2</jats:sub>
) and benzoate dioxygenase of
<jats:italic>Acinetobacter calcoaceticus</jats:italic>
ADP1 (BADO
<jats:sub>ADP1</jats:sub>
) 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
<jats:sub>ADP1</jats:sub>
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
<jats:sub>mt2</jats:sub>
(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
<jats:sub>B</jats:sub>
β
<jats:sub>T</jats:sub>
hybrid oxygenase for these benzoates corresponded to that of BADO
<jats:sub>ADP1</jats:sub>
, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the α
<jats:sub>T</jats:sub>
β
<jats:sub>B</jats:sub>
hybrid oxygenase differed slightly from that of TADO
<jats:sub>mt2</jats:sub>
(3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the α
<jats:sub>T</jats:sub>
β
<jats:sub>B</jats:sub>
hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADO
<jats:sub>mt2</jats:sub>
parent. Finally, the turnover of this
<jats:italic>ortho</jats:italic>
-substituted benzoate was much better coupled to O
<jats:sub>2</jats:sub>
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.
</jats:p> |
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| description | <jats:title>ABSTRACT</jats:title> <jats:p> Toluate dioxygenase of <jats:italic>Pseudomonas putida</jats:italic> mt-2 (TADO <jats:sub>mt2</jats:sub> ) and benzoate dioxygenase of <jats:italic>Acinetobacter calcoaceticus</jats:italic> ADP1 (BADO <jats:sub>ADP1</jats:sub> ) 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 <jats:sub>ADP1</jats:sub> 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 <jats:sub>mt2</jats:sub> (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 <jats:sub>B</jats:sub> β <jats:sub>T</jats:sub> hybrid oxygenase for these benzoates corresponded to that of BADO <jats:sub>ADP1</jats:sub> , the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the α <jats:sub>T</jats:sub> β <jats:sub>B</jats:sub> hybrid oxygenase differed slightly from that of TADO <jats:sub>mt2</jats:sub> (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the α <jats:sub>T</jats:sub> β <jats:sub>B</jats:sub> hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADO <jats:sub>mt2</jats:sub> parent. Finally, the turnover of this <jats:italic>ortho</jats:italic> -substituted benzoate was much better coupled to O <jats:sub>2</jats:sub> 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. </jats:p> |
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| imprint | American Society for Microbiology, 2003 |
| imprint_str_mv | American Society for Microbiology, 2003 |
| institution | DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161, DE-Gla1, DE-Zi4, DE-15, DE-Pl11, DE-Rs1, DE-105, DE-14, DE-Ch1, DE-L229 |
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| spelling | Ge, Yong Eltis, Lindsay D. 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.185.18.5333-5341.2003 <jats:title>ABSTRACT</jats:title> <jats:p> Toluate dioxygenase of <jats:italic>Pseudomonas putida</jats:italic> mt-2 (TADO <jats:sub>mt2</jats:sub> ) and benzoate dioxygenase of <jats:italic>Acinetobacter calcoaceticus</jats:italic> ADP1 (BADO <jats:sub>ADP1</jats:sub> ) 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 <jats:sub>ADP1</jats:sub> 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 <jats:sub>mt2</jats:sub> (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 <jats:sub>B</jats:sub> β <jats:sub>T</jats:sub> hybrid oxygenase for these benzoates corresponded to that of BADO <jats:sub>ADP1</jats:sub> , the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the α <jats:sub>T</jats:sub> β <jats:sub>B</jats:sub> hybrid oxygenase differed slightly from that of TADO <jats:sub>mt2</jats:sub> (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the α <jats:sub>T</jats:sub> β <jats:sub>B</jats:sub> hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADO <jats:sub>mt2</jats:sub> parent. Finally, the turnover of this <jats:italic>ortho</jats:italic> -substituted benzoate was much better coupled to O <jats:sub>2</jats:sub> 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. </jats:p> Characterization of Hybrid Toluate and Benzoate Dioxygenases Journal of Bacteriology |
| spellingShingle | Ge, Yong, Eltis, Lindsay D., Journal of Bacteriology, Characterization of Hybrid Toluate and Benzoate Dioxygenases, Molecular Biology, Microbiology |
| title | Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_full | Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_fullStr | Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_full_unstemmed | Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_short | Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| title_sort | characterization of hybrid toluate and benzoate dioxygenases |
| title_unstemmed | Characterization of Hybrid Toluate and Benzoate Dioxygenases |
| topic | Molecular Biology, Microbiology |
| url | http://dx.doi.org/10.1128/jb.185.18.5333-5341.2003 |