Comparative flux control through the cytoplasmic phase of cell wall biosynthesis

Doctoral thesis English OPEN
Bearup, Daniel James
  • Subject: QP

The introduction of antibacterial drugs in the middle of the last century heralded a new era in\ud the treatment of infectious disease. However the parallel emergence of antibiotic resistance and\ud decline in new drug discovery threatens these advances. The development of new antibacterials\ud must therefore be a high priority.\ud The biosynthesis of the bacterial cell wall is the target for several clinically important antibacterials.\ud This extracellular structure is essential for bacterial viability due to its role in the\ud prevention of cell lysis under osmotic pressure. Its principal structural component, peptidoglycan,\ud is a polymer of alternating N-acetyl-glucosamine (GlcNAc) and N-acetyl muramic acid\ud (MurNAc) residues crosslinked by peptide bridges anchored by pentapeptide stems attached\ud to the MurNAc moieties. The biosynthesis of peptidoglycan proceeds in three phases. The\ud first, cytoplasmic, phase is catalysed by six enzymes. It forms a uridine diphosphate (UDP)\ud bound MurNAc residue from UDP-GlcNAc and attaches the pentapeptide stem. This phase is\ud a relatively unexploited target for antibacterials, being targeted by a single clinically relevant\ud antibacterial, and is the subject of this thesis.\ud The Streptococcus pneumoniae enzymes were kinetically characterised and in silico models of\ud this pathway were developed for this species and Escherichia coli. These models were used to\ud identify potential drug targets within each species. In addition the potentially clinically relevant\ud interaction between an inhibitor of and feedback loops within this pathway was investigated.\ud The use of direct parameter estimation instead of more traditional approaches to kinetic characterisation\ud of enzymes was found to have significant advantages where it could be successfully\ud applied. This approach required the theoretical analysis of the models used to determine\ud whether unique parameter vectors could be determined. Such an analysis has been completed\ud for a broad range of biologically relevant enzymes. In addition a relatively new approach to\ud such analysis has been developed.
  • References (60)
    60 references, page 1 of 6

    1.3 In silico modelling of metabolic pathways . . . . . . . . . . . . . . . . . . . . . . 19 1.3.1 Mathematical modelling of metabolism . . . . . . . . . . . . . . . . . . . 20 1.3.2 Kinetic characterisation of enzymes . . . . . . . . . . . . . . . . . . . . . 22 1.3.3 Numerical solution of differential equations . . . . . . . . . . . . . . . . . 23 1.4 Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1.4.1 Outline of the remainder of the thesis . . . . . . . . . . . . . . . . . . . . 24 2.3.10 Coomassier staining of gels . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.3.11 Determination of protein concentration . . . . . . . . . . . . . . . . . . . 35 2.4 Biochemical spectrophotometric assays . . . . . . . . . . . . . . . . . . . . . . . 35 2.4.1 Determination of concentration of substrates . . . . . . . . . . . . . . . . 35 2.4.2 Continuous ADP production assay . . . . . . . . . . . . . . . . . . . . . . 37 2.4.3 Continuous phosphate production assay . . . . . . . . . . . . . . . . . . . 38 2.4.4 Continuous assay of MurB activity . . . . . . . . . . . . . . . . . . . . . 40 2.4.5 Continuous assay of Lactate dehydrogenase activity . . . . . . . . . . . . 41 2.4.6 Determination of pathway fluxes . . . . . . . . . . . . . . . . . . . . . . . 41 2.4.7 Pre-steady state kinetics experiments . . . . . . . . . . . . . . . . . . . . 42

    3. Theoretical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.2 Structural identifiability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.2.1 Taylor series approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3.2.2 Pohjanpalo's Jacobian rank test . . . . . . . . . . . . . . . . . . . . . . . 50 3.2.3 Input-output relationship approach . . . . . . . . . . . . . . . . . . . . . 52 3.2.4 Application of the identifiability analysis techniques to a simple model . 59 3.3 Structural indistinguishability analysis . . . . . . . . . . . . . . . . . . . . . . . 62 3.4 Numerical parameter estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 3.5 Simulation of reaction species concentrations . . . . . . . . . . . . . . . . . . . . 67 3.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

    [124] E Sauvage, F Kerff, M Terrak, J A Ayala, and P Charlier. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev, 32(2):234-58, 2008.

    [125] A Bouhss, M Crouvoisier, D Blanot, and D Mengin-Lecreulx. Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis. J Biol Chem, 279(29):29974-80, 2004.

    [126] S Ha, E Chang, MC Lo, H Men, P Park, M Ge, and S Walker. The kinetic characterization of Escherichia coli MurG using synthetic substrate analogues. J Am Chem Soc, 3:21-28, 1996.

    [127] L Chen, H Men, S Ha, XY Ye, L Brunner, Y Hu, and S Walker. Intrinsic Lipid Preferences and Kinetic Mechanism of Escherichia coli MurG. Biochemistry, 41(21):6824-6833, 2002.

    [128] G Auger, J van Heijenoort, D Mengin-Lecreulx, and D Blanot. A MurG assay which utilises a synthetic analogue of lipid I. FEMS Microbiology Letters, 219(1):115-119, 2003.

    [129] U Kohlrausch, F B Wientjes, and J V Ho¨ltje. Determination of murein precursors during the cell cycle of Escherichia coli. J Gen Microbiol, 135(6):1499-506, 1989.

    [130] R Kopelman. Fractal Reaction Kinetics. Science, 241(4873):1620-1626, 1988.

    [131] JV Holtje. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiology and Molecular Biology Reviews, 62(1):181, 1998.

  • Metrics
    0
    views in OpenAIRE
    0
    views in local repository
    152
    downloads in local repository

    The information is available from the following content providers:

    From Number Of Views Number Of Downloads
    Warwick Research Archives Portal Repository - IRUS-UK 0 152
Share - Bookmark