Informace o publikaci

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

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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.

Rok publikování 2019
Druh Konferenční abstrakty
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Popis 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.