Project information
Structural and biochemical studies of an ancestral enzyme with dual dehalogenase and luciferase activity
(Ancestral)
- Project Identification
- 792772
- Project Period
- 6/2018 - 5/2020
- Investor / Pogramme / Project type
-
European Union
- Horizon 2020
- MSCA Marie Skłodowska-Curie Actions (Excellent Science)
- MU Faculty or unit
- Faculty of Science
Haloalkane dehalogenases (HLDs) catalyse the cleavage of the carbon-halogen bond of industrial organohalogen compounds and are interesting subjects to study molecular evolution. Strikingly, HLDs display remarkable sequence and structural similarity with luciferase from the marine invertebrate Renilla reniformis (RLuc), which reflects their common evolutionary history. Unlike HLDs, which are α/β hydrolases (EC 3.8.1.5), the RLuc luciferase is cofactor-independent monooxygenase (EC 1.13.12.5) that converts coelenterazine into coelenteramide and carbon dioxide, followed by an emission of blue light. Yet, the evolutionary steps driving their functional divergence remain poorly understood. Our proof-of-concept data show the feasibility of the reconstruction of an ancestral enzyme, which existed prior to the functional
divergence of the modern-day HLD and RLuc homologues, and that this in-lab resurrected enzyme exhibits so-far unobserved dual dehalogenase/luciferase activity. This project aims to dissect structural and biochemical basis of this unusual biocatalytic behaviour of the ancestral enzyme. Specifically, X-ray crystallography, including time-resolved studies with photo-switchable substrate analogues, and advanced mass spectrometry techniques will be employed to probe enzyme-substrate complexes in order to get molecular insights into the inner organization and workings of the catalytically promiscuous enzyme. Complementary site-directed mutagenesis and molecular dynamics simulations will explore the contributions of individual amino acid residues to the dual-function activity. The gained knowledge will extend our in-depth understanding of the evolution of underlying biocatalytic reaction mechanisms. Furthermore, it will pave the way for the development of novel software tools for the rational engineering of next-generation biocatalysts for specific uses in biotechnology and biomedicine.
Publications
Total number of publications: 6
2023
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Catalytic mechanism for Renilla-type luciferases
Nature Catalysis, year: 2023, volume: 6, edition: 1, DOI
2021
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Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers
ACS Catalysis, year: 2021, volume: 11, edition: 21, DOI
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Engineering the protein dynamics of an ancestral luciferase
Nature Communications, year: 2021, volume: 12, edition: 1, DOI
2020
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Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
Chemical Science, year: 2020, volume: 11, edition: 41, DOI
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Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family
Computational and Structural Biotechnology Journal, year: 2020, volume: 18, edition: December 2020, DOI
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Structures of hyperstable ancestral haloalkane dehalogenases show restricted conformational dynamics
Computational and Structural Biotechnology Journal, year: 2020, volume: 18, edition: 2020, DOI