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Elucidating dye decolorising peroxidase mechanism by time-resolved electron microscopy

Primary Supervisor: Dr. Hanna Kwon, Institute of Structural and Chemical Biology

Secondary supervisor: Prof. Peter Moody

PhD project title: Elucidating dye decolorising peroxidase mechanism by time-resolved electron microscopy

University of Registration: University of Leicester

Project outline:

In the 20th century, X-ray crystallography revealed a molecular world at a level previously unimaginable, far beyond the limits of the microscope. However, X-ray structures are merely a static “snapshot” representing an ensemble average of rigid macromolecule states. Understanding the structural dynamics of biomolecular catalysis requires atomically resolved structures acquired continuously along an enzyme reaction pathway – a “molecular movie”. One of the few methods that provides this information is time-resolved (tr) crystallography.

In this project, we will study dye decolorising peroxidases (DyPs) via time-resolved techniques including x-ray crystallography and cryo-EM. DyPs are the most recent member of the peroxidase family to be discovered and differ significantly in protein fold to their counterparts (e.g., CcP and HRP). DyPs catalyse the oxidation of a wide range of substrates including anthraquinones, which are poor substrates for other peroxidases. Several DyPs can also catalyse some extraordinary reactions such as deferrochelation.

The mechanism of DyPs has been proposed to be similar to that of heme peroxidases, however contradicting literature exists. Furthermore, in contrast to other heme proteins our knowledge about DyPs is very limited. Using time-resolved techniques, we will investigate the mechanisms of DyP proteins to provide much needed structural clarity into deciphering the mechanistic features of this class of enzyme.

The ultimate goal is to obtain the structures with visible hydrogen atoms to create molecular movies of substrate turnover which will enhance our understanding of the catalytic mechanism and the role of conserved residues. To complement this study, neutron crystallography will also be carried out and the crystallography studies will be complemented with UV-vis spectroscopy in crystallo.

DyPs unique substrate specificity and catalytic properties offer great opportunities for exploring peroxidase enzymology and biotechnology application. This study will enhance our understanding of this new superfamily of peroxidases.

BBSRC Strategic Research Priority: Understanding the Rules of Life:Structural Biology

Techniques that will be undertaken during the project:

  • Molecular Biology (cloning & mutagenesis)
  • Protein expression and purification
  • Enzyme kinetics
  • Protein crystallisation
  • Structure determination (X-ray crystallography & cryo-EM)

Contact: Dr Hanna Kwon, University of Leicester