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

a 2019

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

SCHENKMAYEROVA, A., G. PINTO, Martin MAREK, Martin TOUL, Lenka HERNYCHOVÁ et. al.

Základní údaje

Originální název

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

Autoři

SCHENKMAYEROVA, A., G. PINTO, Martin MAREK (203 Česká republika, domácí), Martin TOUL (203 Česká republika, domácí), Lenka HERNYCHOVÁ (203 Česká republika), Veronika LIŠKOVÁ (203 Česká republika, domácí), S. EMOND, David BEDNÁŘ (203 Česká republika, domácí), Zbyněk PROKOP (203 Česká republika, domácí), Radka CHALOUPKOVÁ (203 Česká republika, domácí), F. HOLLFELDER a U.T. BORNSCHEUER

Vydání

European Biotechnology Congress, 2019

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

20800 2.8 Environmental biotechnology

Stát vydavatele

Nizozemské království

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 3.503

Kód RIV

RIV/00216224:14310/19:00113697

Organizační jednotka

Přírodovědecká fakulta

ISSN

DOI

http://dx.doi.org/10.1016/j.jbiotec.2019.05.118

UT WoS

000491118400101

Klíčová slova anglicky

enzyme

Štítky

rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 20. 4. 2020 19:25, Mgr. Marie Šípková, DiS.

Anotace

V originále

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.

Citovat

SCHENKMAYEROVA, A., G. PINTO, Martin MAREK, Martin TOUL, Lenka HERNYCHOVÁ, Veronika LIŠKOVÁ, S. EMOND, David BEDNÁŘ, Zbyněk PROKOP, Radka CHALOUPKOVÁ, F. HOLLFELDER a U.T. BORNSCHEUER. Functional switching based on altered enzyme flexibility via InDel mutagenesis of a reconstructed ancestor. In European Biotechnology Congress. 2019. ISSN 0168-1656. Dostupné z: https://dx.doi.org/10.1016/j.jbiotec.2019.05.118.

@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 a 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. Dostupné z: https://dx.doi.org/10.1016/j.jbiotec.2019.05.118.

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