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
Masarykova univerzita
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.