Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.781886
Title: Investigation of a polyprenyl/dolichyl phosphate mannose synthase involved in bacterial protein mannosylation
Author: Banana-Dube, Pamela
ISNI:       0000 0004 7967 4998
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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Abstract:
Glycosylation is one of the most prevalent post-translational modifications that significantly affects protein structure, localisation and function. Protein mannosylation is particularly crucial for the development of healthy organisms, and it is also involved in the pathogenesis of infectious diseases such as tuberculosis and meningitis. Protein mannosylation is carried out in various organisms by highly specific glycosyltransferases (GTs) that utilise polyprenyl phosphate mannosyl donor substrates, such as undecaprenyl phosphate mannose in prokaryotes and dolichyl phosphate mannose in eukaryotes. This research work concerned the investigation of the function and the structure of polyprenyl/dolichyl phosphate mannose synthase enzymes. These catalyse the conversion of polyprenyl/dolichyl phosphate substrates to the corresponding β-mannosyl phospholipids using GDP-mannose. Currently, there is scarce information about this enzyme class due to challenges associated to protein solubility, stability and limited substrate availability. Insights into the structure and the function of these enzymes would greatly aid the development of glycosyltransferases as both biocatalysts and as novel therapeutics targeting glycosylation machineries. A polyprenyl phosphate mannose synthase, Ppm1, involved in protein glycosylation in the model actinomycete S. coelicolor A3(2), was recombinantly expressed in E. coli in a soluble tagged full-length form and in good yield, as well as a C-terminal truncated version (∆42 Ppm1). The proteins were purified by nickel affinity and gel filtration in the presence of various detergents, from which it emerged that the enzymes are present in dimeric forms, albeit at high concentrations formation of trimers and further multimers occur. A robust assay protocol based on a coupled phosphatase reaction was utilised to evaluate the activity of Ppm1, which exhibited promiscuity for both donor nucleotide substrates and acceptor lipid phosphates of varying chain lengths and degrees of saturation. Kinetic parameters, KM and kcat, of Ppm1 were also determined, with the enzyme displaying better catalytic efficiency for the donor substrate GDP-mannose compared to the acceptor substrate undecaprenyl phosphate. To further investigate the enzyme structural requirements for biocatalysis, site-directed mutagenesis was employed to generate several enzyme mutants. These were successfully expressed in E. coli, purified and subsequently utilised for functional assays. The results of these functional assays revealed which residues are important for catalysis and helped us to build a substrate binding model for Ppm1. This information was further substantiated by collaborative studies looking at antibiotic hypersensitivity in S. coelicolor itself. In this work we also report the first evidence of chemical rescue for an inverting glycosyltransferase family 2 (GT2) enzyme: indeed, the activity of one of the Ppm1 mutants that mimic a defective human DPM1 synthase, R82A, was successfully rescued in vitro using exogenous small molecules. Enzyme crystallisation trials of Ppm1 along with several mutants, the truncated enzyme and a thermostable homologue from Streptomyces thermolilacinus were initiated, alongside solution NMR studies to investigate the enzyme behaviour and dynamic structure in solution. Encouraging preliminary results suggest that in the near future it should be possible to solve the enzyme structure and obtain ligand-enzyme binding affinities (Kd values) for various acceptor and donor substrates, as well as inhibitors, via NMR.
Supervisor: Not available Sponsor: University of Warwick
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.781886  DOI: Not available
Keywords: QD Chemistry ; QP Physiology
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