Functional and structural characterisation of glycosyltransferases involved in protein mannosylation

Secondary Supervisor(s): Professor Alexander Cameron and Dr Deborah Brotherton

University of Registration: University of Warwick

BBSRC Research Themes:

• Sustainable Agriculture and Food (Animal Health and Welfare)
• Understanding the Rules of Life (Immunology, Microbiology, Stem Cells, Structural Biology)
• Integrated Understanding of Health (Diet and Health, Pharmaceuticals)

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, membrane-bound glycosyltransferases that utilise complex mannosyl donors. This membrane association means 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 structure and function of key glycosyltransferases involved in the mannosylation of proteins and small bioactive molecules in bacteria and humans.

The project work will include gene cloning, protein expression in heterologous hosts, protein purification, site-directed mutagenesis and the chemoenzymatic preparation of sugar donors/substrates for the functional and structural characterisation of these enzymes via kinetics, crystallisation and cryoEM.

The results from these experiments will provide crucial insights on mannosylation, with important implications for the understanding of prokaryotic and eukaryotic biology and future drug/therapy design targeting glycosylation machineries.

For further project details please contact m.tosin@warwick.ac.uk.

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.

2. M. Lommel, S. Strahl, ‘Protein O-mannosylation: conserved from bacteria to humans’, Glycobiology 2009, 19, 816.

3. 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.

4. J. Hang et al., ‘Protein O-mannosylation across kingdoms and related diseases: From glycobiology to glycopathology’, Biomed. Pharmacother. 2022, 148, 112685.

Novel Chemoenzymatic Approaches to ‘Unnatural’ Products

Secondary Supervisor(s): Dr Christopher Corre

University of Registration: University of Warwick

BBSRC Research Themes:

• Renewable Resources and Clean Growth (Bio-energy, Industrial Biotechnology)
• Understanding the Rules of Life (Microbiology)
• Integrated Understanding of Health (Diet and Health, Pharmaceuticals)

Project Outline

Natural products represent a major source of powerful and innovative therapeutic agents for the treatment of human, animal and plant diseases.¹ Many of these products, including broad-spectrum and last resort antibiotics such as erythromycin, vancomycin and taxol are of polyketide, nonribosomal peptide and terpene origin. The structural complexity of these molecules still provides stimulating and challenging targets for synthetic organic chemists. Moreover, synthetic biology is currently seen as a highly attractive avenue for complex natural product preparation as well as structural diversification.² This latter is highly desirable in view of developing new bioactive molecules of increased efficiency and/or novel activities.³

Polyketide and nonribosomal peptide products are biosynthesized in microorganisms and plants by multifunctional enzymes, the polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) respectively. These utilize simple and abundant building blocks (e.g. acyl and amino acyl) and iterative condensation reactions to generate highly chemically functionalised polyketide/peptide chains, which can be further enzymatically tailored to yield the final natural products.^{4, 5} In the Tosin group at Warwick Chemistry we have found that small synthetic molecules that mimic the building blocks utilized in polyketide and nonribosomal peptide assembly can react with ‘stalled’ enzyme-bound biosynthetic intermediates and lead to the formation of ‘unnatural’ products: ^6-8 these are structurally complex molecules that are still made by a PKS enzymatic assembly but that are structurally different from the original products. Unnatural products may be as medicinally/commercially valuable as existing natural products, if not superior.^{3, 6}

The overall aim of this PhD project is to generate novel unnatural products from simple synthetic precursors (‘synthons’) and genetically engineered bacteria. The following specific research objectives will be pursued:

(i) the preparation of a library of novel chemical synthons bearing functionalities that cannot be made biosynthetically (organic synthesis, under the supervision of Dr Manuela Tosin);

(ii) the genetic engineering of Streptomyces bacteria to allow/facilitate the chemical synthons cellular uptake and their incorporation into selected polyketide/ nonribosomal peptide/terpene pathways (molecular biology, under the supervision of Dr Christophe Corre);

(iii) the generation of unnatural products in vitro and in vivo, by providing enzymes/ engineered bacterial cells with the chemical synthons, followed by product isolation and characterization (microbiology and analytical chemistry, under the supervision of Dr Manuela Tosin). Preliminary bioassays of the novel products will be undertaken, either in the host lab(s) or through collaborations.

References

1. D. J. Newman et al., J. Nat. Prod. 2012, 75, 311.

2. M. R. Seyedsayamdost et al., ACS Synth. Biol.2014, 3, 745.

3. R. J. M. Goss et al., Nat. Prod. Rep. 2012, 29, 870-889.

4. K. J. Weissman, Methods Enzymol. 2009, 459, 3-16.

5. E. A. Felnagle et al., Mol. Pharmaceutics 2008, 5, 191- 211.

6. E. Riva et al., Angew. Chem. Int. Ed. 2014 ,53, 11944-9 (VIP article).

7. C. H. Y. Ho et al., Chem. Commun. 2017, 53, 7088.

8. D. J. Leng et al., Chem. Commun. 2021, 5, 2293.

A fragment-based approach towards novel agents against the fungal superbug Candida auris

Secondary Supervisor(s): Dr Phillip Stansfeld

University of Registration: University of Warwick

BBSRC Research Themes:

• Understanding the Rules of Life (Immunology, Microbiology, Structural Biology)
• Integrated Understanding of Health (Pharmaceuticals)

Project Outline

Over the past 15 years, Candida auris has emerged as one of the most feared hospital microbes.¹ This yeast-type fungus most frequently causes bloodstream infections and can also affect the respiratory system, the central nervous system, internal organs and the skin, with mortality rates ranging from 30% to 72% for patients affected by invasive C. auris infections. The latter mostly occur in care settings (e.g. in hospitals) and are often misdiagnosed; worryingly, the majority of C. auris strains are resistant to the main classes of antifungal drugs currently in use, such as fluconazole, amphotericin and echinocandins.² Hence, there is an urgent need to identify new targets and develop new molecules to tackle life-threatening and multi-drug-resistant C. auris infections.

In collaboration with the group of Dr Marcio Dias (Department of Microbiology, Institute of Biomedical Science, Sao Paulo, Brazil) we have recently contributed to the structural and functional characterisation of C. auris dihydrofolate reductase (DHFR).³ DHFR is an essential enzyme for DNA synthesis and processing, and it constitutes an effective therapeutic target for other Candida species and beyond.⁴ We have also preliminarily identified promising small molecule ‘fragments’ capable of binding to and inhibiting C. auris DHRF (unpublished work).

The aim of this PhD project is to develop novel antifungal agents targeting C. auris DHFR. The project work will include: the synthesis of a fragment- based library of compounds aimed at probing and ultimately inhibiting C. auris DHFR function (under the supervision of Dr Tosin, Warwick Chemistry); molecular docking and molecular dynamics to aid and guide compound design (under the supervision of Prof Phill Stansfeld, School of Life Sciences); recombinant protein expression and purification, DHFR activity assays in the presence of the newly synthesised compounds (at Warwick Chemistry); compound co-crystallisation with C. auris DHFR and activity evaluation against C. auris strains (at Sao Paulo, under the supervision of Dr Marcio Dias).

The results of this project will contribute to the understanding of pathogen behaviour/targets and pathogen/host interactions, as well as to the development of novel pharmaceuticals tacking a global and immediate medical need.

For further project details please contact m.tosin@warwick.ac.uk.

References

1. WHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action, 2022.

2. Jie, T. et al. Acta Pharmaceutica Sinica B 2022, 12, 4056.

3. Kirkman, T. et al. Acta Crystallogr. D 2023, 79, 735.

4. Wróbel, A. et al. J. Antibiot. 2020, 73, 5.