Informace o projektu
Decoding the molecular principles of enzyme evolution
- Kód projektu
- MUNI/H/1561/2018
- Období řešení
- 1/2019 - 12/2021
- Investor / Programový rámec / typ projektu
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Masarykova univerzita
- Grantová agentura MU
- Individuální projekty hraničního výzkumu
- Fakulta / Pracoviště MU
- Přírodovědecká fakulta
Molecular evolution is one of the most important hallmarks of biology. Gain of catalytic functions at the enzyme class level provides unique opportunity to track evolutionary pathways leading to novel biological functions. Our model enzymes haloalkane dehalogenases catalyse the cleavage of the carbon-halogen bond of organohalogen compounds. Strikingly, haloalkane dehalogenases display remarkable sequence and structural similarity with light-emitting luciferase from the marine invertebrate Renilla reniformis, reflecting their common evolutionary history. Unlike haloalkane dehalogenases, which are α/β hydrolases (EC 3.8.1.5), the Renilla 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. 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 enzymes, and this in-lab resurrected enzyme exhibits so-far unobserved dual dehalogenase/luciferase activity. This high-risk project has an ambition to dissect structural and biochemical basis of this unusual biocatalytic behaviour of the ancestral enzyme, by an integrated structural and chemical biology approach. 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 to get molecular insights into the inner organization of the catalytically promiscuous enzyme. Site-directed mutagenesis and molecular dynamics simulations will explore the contributions of individual amino acid residues to the dual activity. The gained knowledge will extend our in-depth understanding of the evolution beyond the state-of-the-art, particularly by unravelling key structural and dynamical determinants that dictated functional divergence of enzymes. High-gain and high-impact innovation of this project will pave the way for the development of new theoretical concepts and cutting-edge software tools for the rational engineering of next-generation biocatalysts for biotechnology and biomedicine.
Publikace
Počet publikací: 9
2023
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Catalytic mechanism for Renilla-type luciferases
Nature Catalysis, rok: 2023, ročník: 6, vydání: 1, DOI
2022
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Extended Mechanism of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis: 1000-fold Higher Catalytic Activity than That of Clinically Used Alteplase
ACS Catalysis, rok: 2022, ročník: 12, vydání: 7, DOI
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Increased occurrence of Treponema spp. and double-species infections in patients with Alzheimer's disease
Science of the Total Environment, rok: 2022, ročník: 844, vydání: October 2022, DOI
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Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling
JACS AU, rok: 2022, ročník: 2, vydání: 6, DOI
2021
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Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers
ACS Catalysis, rok: 2021, ročník: 11, vydání: 21, DOI
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Engineering the protein dynamics of an ancestral luciferase
Nature Communications, rok: 2021, ročník: 12, vydání: 1, DOI
2020
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Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
Chemical Science, rok: 2020, ročník: 11, vydání: 41, DOI
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Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family
Computational and Structural Biotechnology Journal, rok: 2020, ročník: 18, vydání: December 2020, DOI
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Structures of hyperstable ancestral haloalkane dehalogenases show restricted conformational dynamics
Computational and Structural Biotechnology Journal, rok: 2020, ročník: 18, vydání: 2020, DOI