Skip to main content

Functional and structural characterization of a glycosyltransferase involved in bacterial mannosylation

Principal Supervisor: Dr Manuela Tosin - Department of Chemistry

Co-supervisors: Dr Alex Cameron - School of Life Sciences and Dr Józef Lewandowski - Department of Chemistry

PhD project title: Functional and structural characterization of a glycosyltransferase involved in bacterial mannosylation

University of Registration: Warwick

Project outline:

Glycosylation is one the most abundant post-translational modifications and an evolutionary indicator: indeed the increasing complexity of host-pathogen and organism-environment interactions, amongst others, is reflected in the newly-reported ability of prokaryotes (e.g. bacteria and archaea) to carry out eukaryotic functions, such as glycosylation of proteins and other biomolecules.1-2

Protein mannosylation is vital for healthy organisms3 and is also involved in the pathogenesis of many virulent diseases, such as meningitis and tubercolosis.4 It is carried out in different organisms by highly specific glycosyltransferases that utilise complex mannosyl donors and are generally membrane-bound; therefore there is still a general lack of direct information on the enzyme catalysis and structure.

The aim of this PhD project is to fully elucidate the function and the structure of a key bacterial glycosyltransferase involved in the mannosylation of proteins and small bioactive molecules.

The project work will include gene cloning, enzyme expression and purification, site-directed mutagenesis and the chemoenzymatic preparation of sugar donors/substrates for the enzyme functional and structural characterisation via kinetics, crystallisation and NMR spectroscopy investigations.

The results from these experiments will give for the first time a full picture of a key enzyme involved in protein mannosylation, with important implications for the understanding of prokaryotic and eukaryotic glycosylation and future drug/therapy design targeting glycosylation machineries.

References:

  1. M. Abu-Qarn, J. Eichler, N. Sharon, ‘Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea’, Curr Opin Struct Biol. 2008, 18, 544-50.
  2. C. Espitita, L. Servin-Gonzales, R. Mancilla, ‘New insights into protein O-mannosylation in actinomycetes’, Mol. BioSyst. 2010, 6, 775-781.
  3. M. Lommel, S. Strahl, ‘Protein O-mannosylation: conserved from bacteria to humans’, Glycobiology 2009, 19, 816-828.
  4. C.-F. Liu et al., ‘Bacterial protein O-mannosylating enzyme is crucial for virulence of Mycobacterium tubercolosis’, Proc Natl Acad Sci USA 2013, 110, 6560-6565.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Molecular biology for gene cloning and site-directed mutagenesis: PCR, digestion of DNA with restriction enzymes, agarose gel electrophoresis
  • Protein expression and purification by affinity chromatography, size-exclusion, ion- exchange, hydrophobic interactions
  • Enzymology: enzyme activity assays with natural and synthetic substrates; enzyme kinetics (UV, HPLC, ..)
  • Small molecules, DNA and protein characterisation via a range of analytical techniques, including mass spectrometry, fluorescence, circular dichroism, electrophoresis
  • Chemoenzymatic preparation of carbohydrate donors/substrates
  • Protein X-ray crystallography (at the Warwick Macromolecular Crystallisation Facility, Life Sciences): sample preparation, set up of automated crystallisation screening (using the Honeybee 963 nanodrop crystallisation robot), crystal growth optimisation, X-ray diffraction patterns analyses and protein structure resolution
  • Protein NMR: spectral assignment in solution and solid state, chemical shift perturbations, structure determination both in solution and solid-state, protein dynamics in solution and solid-state
  • Bioinformatics: design of PCR primers, analysis of DNA/protein sequences, protein structural modelling and analyses

Contact: Dr Manuela Tosin, University of Warwick