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@article{1403840,
author = {Babková, Petra and Šebestová, Eva and Brezovský, Jan and Chaloupková, Radka and Damborský, Jiří},
article_number = {14},
doi = {http://dx.doi.org/10.1002/cbic.201700197},
keywords = {ancestral sequence reconstruction; haloalkane dehalogenase; protein engineering; robustness; substrate specificity},
language = {eng},
issn = {1439-4227},
journal = {CHEMBIOCHEM},
title = {Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity},
url = {https://loschmidt.chemi.muni.cz/peg/publications/ancestral-haloalkane-dehalogenases-show-robustness-and-unique-substrate-specificity/},
volume = {18},
year = {2017}
}
TY - JOUR
ID - 1403840
AU - Babková, Petra - Šebestová, Eva - Brezovský, Jan - Chaloupková, Radka - Damborský, Jiří
PY - 2017
TI - Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity
JF - CHEMBIOCHEM
VL - 18
IS - 14
SP - 1448-1456
EP - 1448-1456
SN - 14394227
KW - ancestral sequence reconstruction
KW - haloalkane dehalogenase
KW - protein engineering
KW - robustness
KW - substrate specificity
UR - https://loschmidt.chemi.muni.cz/peg/publications/ancestral-haloalkane-dehalogenases-show-robustness-and-unique-substrate-specificity/
N2 - Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1–5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (DTm up to 24 8C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.
ER -
BABKOVÁ, Petra, Eva ŠEBESTOVÁ, Jan BREZOVSKÝ, Radka CHALOUPKOVÁ and Jiří DAMBORSKÝ. Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity. \textit{CHEMBIOCHEM}. 2017, vol.~18, No~14, p.~1448-1456. ISSN~1439-4227. Available from: https://dx.doi.org/10.1002/cbic.201700197.
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