Engineering the protein dynamics of an ancestral luciferase

Andrea Schenkmayerova, Gaspar P. Pinto, Martin Toul, Martin Marek, Lenka Hernychova, Joan Planas-Iglesias, Veronika Daniel Liskova, Daniel Pluskal, Michal Vasina, Stephane Emond, Mark Dörr, Radka Chaloupkova, David Bednar, Zbynek Prokop, Florian Hollfelder (), Uwe T. Bornscheuer () and Jiri Damborsky ()
Additional contact information
Andrea Schenkmayerova: St. Anne’s University Hospital Brno
Gaspar P. Pinto: St. Anne’s University Hospital Brno
Martin Toul: St. Anne’s University Hospital Brno
Martin Marek: Masaryk University
Lenka Hernychova: Masaryk Memorial Cancer Institute
Joan Planas-Iglesias: St. Anne’s University Hospital Brno
Veronika Daniel Liskova: St. Anne’s University Hospital Brno
Daniel Pluskal: Masaryk University
Michal Vasina: St. Anne’s University Hospital Brno
Stephane Emond: University of Cambridge
Mark Dörr: University of Greifswald
Radka Chaloupkova: Masaryk University
David Bednar: St. Anne’s University Hospital Brno
Zbynek Prokop: St. Anne’s University Hospital Brno
Florian Hollfelder: University of Cambridge
Uwe T. Bornscheuer: University of Greifswald
Jiri Damborsky: St. Anne’s University Hospital Brno

Nature Communications, 2021, vol. 12, issue 1, 1-16

Abstract: Abstract Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

Date: 2021
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-23450-z

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DOI: 10.1038/s41467-021-23450-z

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