Functional switching based on altered enzyme flexibility via InDel mutagenesis of a reconstructed ancestor

Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F19%3A00113697" target="_blank" >RIV/00216224:14310/19:00113697 - isvavai.cz</a>
Result on the web
<a href="https://doi.org/10.1016/j.jbiotec.2019.05.118" target="_blank" >https://doi.org/10.1016/j.jbiotec.2019.05.118</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1016/j.jbiotec.2019.05.118" target="_blank" >10.1016/j.jbiotec.2019.05.118</a>

Result language
angličtina
Original language name
Functional switching based on altered enzyme flexibility via InDel mutagenesis of a reconstructed ancestor
Original language description
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.
Czech name
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Czech description
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Project
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Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year
2019
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

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