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An investigation into an unusual glycan branching enzyme from Mycobacterium tuberculosis

An investigation into an unusual glycan branching enzyme from Mycobacterium tuberculosis
An investigation into an unusual glycan branching enzyme from Mycobacterium tuberculosis
Mycobacterium tuberculosis (Mtb), the pathogen that causes tuberculosis (TB), is showing increasing resistance to current drug therapies. It is therefore essential that novel drug targets and drug therapies are discovered. Methylglucose lipopolysaccharides (MGLPS) are glycoconjugates produced by Mycobacteria identified as essential for viability. The genes involved in the biosynthesis of MGLPs have been shown to be essential for the survival of the bacterium. Initial stages of the biosynthetic pathway to MGLPs are thought to include the transfer of a glucose unit to glucosyl glycerate (GG), forming di glucosyl glycerate. The gene responsible for this reaction has been identified as Rv3031 and is thought to encode a GH57 glucan branching enzyme (GBE). Known enzymes belonging to this family act upon large substrates, however, the proposed biosynthetic pathway to MGLPs implies that the MtGBE works on much smaller carbohydrate units.

The full chemical synthesis of several carbohydrate compounds was performed, including the synthesis of GG and a novel synthetic route to maltosyl glycerate (MalG). Compounds were characterised using NMR spectroscopy and mass spectrometry and GG and MalG were tested in enzymatic assays designed to identify the substrates of MtGBE and MtGlcT.

The co-expression of MtGBE with the chaperone system GroEL-GroES is described, as well as the purification of the protein by Ni-NTA affinity column. Various assay techniques were utilised to investigate the nature of the activity of MtGBE and to identify the glycan donor. The glycosyl transferase encoded for by Rv3032 was expressed and isolated from inclusion bodies to investigate potential co-operation between this and MtGBE. Though no hydrolysis or branching activity was observed and no donors identified, this is the first time MtGBE protein has been expressed and studied. This work can rule out certain types of activity thus clearing the way to discovering the true nature of the MtGBE enzyme.
University of Southampton
Gusthart, Jessica Sophie
284bc0bc-e861-4059-98dd-c658e4d86acc
Gusthart, Jessica Sophie
284bc0bc-e861-4059-98dd-c658e4d86acc
Lee, Seung
ee34fa26-5fb6-48c8-80c2-1f13ec4ccceb

Gusthart, Jessica Sophie (2018) An investigation into an unusual glycan branching enzyme from Mycobacterium tuberculosis. University of Southampton, Doctoral Thesis, 370pp.

Record type: Thesis (Doctoral)

Abstract

Mycobacterium tuberculosis (Mtb), the pathogen that causes tuberculosis (TB), is showing increasing resistance to current drug therapies. It is therefore essential that novel drug targets and drug therapies are discovered. Methylglucose lipopolysaccharides (MGLPS) are glycoconjugates produced by Mycobacteria identified as essential for viability. The genes involved in the biosynthesis of MGLPs have been shown to be essential for the survival of the bacterium. Initial stages of the biosynthetic pathway to MGLPs are thought to include the transfer of a glucose unit to glucosyl glycerate (GG), forming di glucosyl glycerate. The gene responsible for this reaction has been identified as Rv3031 and is thought to encode a GH57 glucan branching enzyme (GBE). Known enzymes belonging to this family act upon large substrates, however, the proposed biosynthetic pathway to MGLPs implies that the MtGBE works on much smaller carbohydrate units.

The full chemical synthesis of several carbohydrate compounds was performed, including the synthesis of GG and a novel synthetic route to maltosyl glycerate (MalG). Compounds were characterised using NMR spectroscopy and mass spectrometry and GG and MalG were tested in enzymatic assays designed to identify the substrates of MtGBE and MtGlcT.

The co-expression of MtGBE with the chaperone system GroEL-GroES is described, as well as the purification of the protein by Ni-NTA affinity column. Various assay techniques were utilised to investigate the nature of the activity of MtGBE and to identify the glycan donor. The glycosyl transferase encoded for by Rv3032 was expressed and isolated from inclusion bodies to investigate potential co-operation between this and MtGBE. Though no hydrolysis or branching activity was observed and no donors identified, this is the first time MtGBE protein has been expressed and studied. This work can rule out certain types of activity thus clearing the way to discovering the true nature of the MtGBE enzyme.

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JESSICA GUSTHART FINAL THESIS - Version of Record
Available under License University of Southampton Thesis Licence.
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Published date: October 2018

Identifiers

Local EPrints ID: 425868
URI: http://eprints.soton.ac.uk/id/eprint/425868
PURE UUID: 97660510-6c44-45e2-8458-3bef23c03676
ORCID for Seung Lee: ORCID iD orcid.org/0000-0002-8598-3303

Catalogue record

Date deposited: 05 Nov 2018 17:30
Last modified: 14 Mar 2019 01:35

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