Alteration of the Substrate Specificity of Benzoylformate Decarboxylase from Pseudomonas putida by Directed Evolution

ChemBioChem ◽  
2003 ◽  
Vol 4 (8) ◽  
pp. 721-726 ◽  
Author(s):  
Bettina Lingen ◽  
Doris Kolter-Jung ◽  
Pascal Dünkelmann ◽  
Ralf Feldmann ◽  
Joachim Grötzinger ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Directed Evolution ◽  
Benzoylformate Decarboxylase

scholarly journals Characterization of Hybrid Toluate and Benzoate Dioxygenases

2003 ◽  
Vol 185 (18) ◽  
pp. 5333-5341 ◽  
Author(s):  
Yong Ge ◽  
Lindsay D. Eltis
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Acinetobacter Calcoaceticus ◽  
The Other ◽  
Β Subunit ◽  
Structural Determinants ◽  
Α Subunit ◽  
Benzoate Dioxygenase

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) 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 BADOADP1 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 TADOmt2 (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 αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 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 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.

Directed Evolution of an Amine Oxidase Possessing both Broad Substrate Specificity and High Enantioselectivity

2003 ◽  
Vol 42 (39) ◽  
pp. 4807-4810 ◽  
Author(s):  
Reuben Carr ◽  
Marina Alexeeva ◽  
Alexis Enright ◽  
Tom S. C. Eve ◽  
Michael J. Dawson ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Directed Evolution ◽  
Amine Oxidase ◽  
Broad Substrate Specificity

scholarly journals Poly-3-hydroxyalkanoate synthases from Pseudomonas putida U: substrate specificity and ultrastructural studies

2007 ◽  
Vol 1 (2) ◽  
pp. 170-176 ◽  
Author(s):  
Sagrario Arias ◽  
Angel Sandoval ◽  
Mario Arcos ◽  
Librada M. Cañedo ◽  
Beatriz Maestro ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Ultrastructural Studies

scholarly journals Altering the Substrate Specificity of Cephalosporin Acylase by Directed Evolution of the β-Subunit

2002 ◽  
Vol 277 (44) ◽  
pp. 42121-42127 ◽  
Author(s):  
Linda G. Otten ◽  
Charles F. Sio ◽  
Johanna Vrielink ◽  
Robbert H. Cool ◽  
Wim J. Quax
Keyword(s):  
Substrate Specificity ◽  
Directed Evolution ◽  
Β Subunit ◽  
Cephalosporin Acylase

Altering the Substrate Specificity of RhlI by Directed Evolution

ChemBioChem ◽  
2009 ◽  
Vol 10 (3) ◽  
pp. 553-558 ◽  
Author(s):  
Pavan Kumar Reddy Kambam ◽  
Dawn T. Eriksen ◽  
Jason Lajoie ◽  
Daniel J. Sayut ◽  
Lianhong Sun
Keyword(s):  
Substrate Specificity ◽  
Directed Evolution
Sign in / Sign up

Export Citation Format

Share Document