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@proceedings{1647638,
author = {Schenkmayerova, A. and Pinto, G. and Marek, Martin and Toul, Martin and Hernychová, Lenka and Lišková, Veronika and Emond, S. and Bednář, David and Prokop, Zbyněk and Chaloupková, Radka and Hollfelder, F. and Bornscheuer, U.T.},
booktitle = {European Biotechnology Congress},
doi = {http://dx.doi.org/10.1016/j.jbiotec.2019.05.118},
keywords = {enzyme},
language = {eng},
title = {Functional switching based on altered enzyme flexibility via InDel mutagenesis of a reconstructed ancestor},
url = {https://doi.org/10.1016/j.jbiotec.2019.05.118},
year = {2019}
}
TY - CONF
ID - 1647638
AU - Schenkmayerova, A. - Pinto, G. - Marek, Martin - Toul, Martin - Hernychová, Lenka - Lišková, Veronika - Emond, S. - Bednář, David - Prokop, Zbyněk - Chaloupková, Radka - Hollfelder, F. - Bornscheuer, U.T.
PY - 2019
TI - Functional switching based on altered enzyme flexibility via InDel mutagenesis of a reconstructed ancestor
KW - enzyme
UR - https://doi.org/10.1016/j.jbiotec.2019.05.118
L2 - https://doi.org/10.1016/j.jbiotec.2019.05.118
N2 - We have resurrected a bifunctional ancestral enzyme that existed prior to the functional diversification into haloalkane dehalogenases (EC 3.8.1.5) and light emitting Renilla luciferase (EC 1.13.12.5). This ancestor, which exhibited markedly enhanced thermal stability, was subjected to InDel mutagenesis to uncover molecular determinants important for the evolution of luciferase activity. Generated libraries were screened and the best hits carrying alterations in three hot-spot regions were characterized. Unexpectedly, the most potent hits contained insertion/substitution events in a most flexible region of the cap domain, as evidenced by biochemical and structural analyses. Indication that protein conformational dynamics plays an important role in luciferase reaction was further supported by molecular dynamics simulations, hydrogen-deuterium exchange analysis and transient kinetics. Collectively, we reveal molecular determinants required for evolvability of luciferase activity and propose a new design to switch enzyme functions by engineering of flexible elements.
ER -
SCHENKMAYEROVA, A., G. PINTO, Martin MAREK, Martin TOUL, Lenka HERNYCHOVÁ, Veronika LIŠKOVÁ, S. EMOND, David BEDNÁŘ, Zbyněk PROKOP, Radka CHALOUPKOVÁ, F. HOLLFELDER and U.T. BORNSCHEUER. Functional switching based on altered enzyme flexibility via InDel mutagenesis of a reconstructed ancestor. In \textit{European Biotechnology Congress}. 2019. ISSN~0168-1656. Available from: https://dx.doi.org/10.1016/j.jbiotec.2019.05.118.
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