Skip to main content

Structural and biochemical studies of an ancestral enzyme with dual dehalogenase and luciferase activity

Objective

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.

Field of science

  • /natural sciences/earth and related environmental sciences/geology/mineralogy/crystallography
  • /natural sciences/chemical sciences/inorganic chemistry/inorganic compounds
  • /natural sciences/chemical sciences/organic chemistry/organohalogen compounds
  • /natural sciences/computer and information sciences/software
  • /natural sciences/chemical sciences/organic chemistry/amines
  • /natural sciences/biological sciences/molecular biology/molecular evolution
  • /humanities/history and archaeology/history
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins/enzymes
  • /natural sciences/chemical sciences/analytical chemistry/mass spectrometry

Call for proposal

H2020-MSCA-IF-2017
See other projects for this call

Funding Scheme

MSCA-IF-EF-ST - Standard EF

Coordinator

Masarykova univerzita
Address
Zerotinovo Namesti 9
60177 Brno Stred
Czechia
Activity type
Higher or Secondary Education Establishments
EU contribution
€ 142 720,80