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

scholarly journals Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Gordon Rix ◽  
Ella J. Watkins-Dulaney ◽  
Patrick J. Almhjell ◽  
Christina E. Boville ◽  
Frances H. Arnold ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Thermotoga Maritima ◽  
Enzyme Engineering ◽  
Tryptophan Synthase ◽  
Β Subunit ◽  
Evolutionary Processes ◽  
Separate Species ◽  
Continuous Evolution ◽  
Primary Activity ◽  
Natural Diversity

Abstract Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing l-tryptophan from indole and l-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

scholarly journals Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

2015 ◽  
Vol 112 (47) ◽  
pp. 14599-14604 ◽  
Author(s):  
Andrew R. Buller ◽  
Sabine Brinkmann-Chen ◽  
David K. Romney ◽  
Michael Herger ◽  
Javier Murciano-Calles ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Pyrococcus Furiosus ◽  
Tryptophan Synthase ◽  
Β Subunit ◽  
Allosteric Activation ◽  
Α Subunit ◽  
X Ray ◽  
Catalytic Activities ◽  
Catalytic Potential ◽  
Protein Partners

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.

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

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

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
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