Biochemical characterisation of pivotal enzymes involved in Mycobacterium tuberculosis cell wall biosynthesis

Harrison, James (2016). Biochemical characterisation of pivotal enzymes involved in Mycobacterium tuberculosis cell wall biosynthesis. University of Birmingham. Ph.D.

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Abstract

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The mycobacterial cell wall consists of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are adorned with a complex polysaccharide, arabinogalactan (AG), via a unique α-L-rhamnopyranose–(1→3)-α-D-GlcNAc-(1→P) linker unit. Whilst the cytoplasmic steps of mycobacterial cell wall biosynthesis have been largely delineated, the molecular processes that govern the flux of PG intermediates and the mechanism by which PG and AG pathways converge has remained elusive. We identified key conserved serine/threonine residues of MurC, as potential candidates for phospho-regulation by the cognate protein kinase, PknA. Pseudo-phosphorylated MurC mutants exhibited differential enzyme activity, suggesting that M. tuberculosis is capable of tight control of PG biosynthesis through phosphorylation of MurC. In addition, we have identified Lcp1, a mycobacterial orthologue of the LytR-CpsA-Psr (LCP) family of proteins found in Gram-positive bacteria, responsible for ligating cell wall teichoic acids to PG. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is a phosphotransferase capable of ligating AG to PG in a cell free radiolabelled assay.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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