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See detailStructural and biochemical study of the proteins AmiC, NlpD and FtsW involved in the bacterial cell division
Rocaboy, Mathieu ULg

Doctoral thesis (2013)

Cell division in the Gram negative bacterium Escherichia coli is a highly coordinated mechanism involving various physiological functions such as chromosome segregation, cell envelope invagination ... [more ▼]

Cell division in the Gram negative bacterium Escherichia coli is a highly coordinated mechanism involving various physiological functions such as chromosome segregation, cell envelope invagination, peptidoglycan synthesis at the division site and separation of the daughter cells. All these functions require a high level of spatio-temporal regulation in order to preserve the physical integrity of the cell. At least 20 proteins required for a proper cell division are recruited to the division site to form a supramolecular complex called the divisome. This thesis work focused on three major components of the E. coli division machinery: the N-acetylmuramyl L-alanine amidase AmiC, the LytM factor NlpD and the lipid II flippase FtsW. These proteins are recruited at midcell at a late stage of cell division. FtsW is an integral membrane protein crucial for the translocation of the peptidoglycan precursor from the cytoplasm to the periplasm where it will be processed to produce septal peptidoglycan. AmiC acts as a septal peptidoglycan hydrolase that allow the separation of the daughter cells. This enzyme has been shown to be activated by the LytM factor NlpD. The crystal structure of AmiC from E. coli presented in this work confirms the presence of an inhibitory helix in the active site. The AmiC variant lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. Furthermore, the direct interaction between AmiC and NlpD has been detected by microscale thermophoresis with an apparent Kd of about 13 µM. The crystal structure of AmiC also reveals the β-sandwich fold of the AMIN domain, responsible for the septal targeting of AmiC to the division site. The two symmetrical four-stranded β-sheets exhibit highly conserved motifs on the two outer faces. Along with the peptidoglycan binding capacity of the AMIN domain, results obtained so far suggest that the AMIN domain could be involved in the recognition of a specific peptidoglycan architecture or a composition different than the lateral peptidoglycan. Production screenings of FtsW from different strains were realized and FstW from E. coli was purified. This challenging project will require additional efforts to obtain sufficient amount of protein for structural investigation. Information gathered in this work confirms the high level of regulation of the hydrolytic activity at the septum and gives a structural basis for a more precise molecular characterization of the division site targeting. Disruption or over-activation of these regulation mechanisms could represent a new strategy in the development of antibacterial compounds. [less ▲]

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See detailCrystal structure of a complex between the Actinomadura R39 DD-peptidase and a peptidoglycan-mimetic boronate inhibitor: interpretation of a transition state analogue in terms of catalytic mechanism.
Dzhekieva, Liudmila; Rocaboy, Mathieu ULg; Kerff, Frédéric ULg et al

in Biochemistry (2010), 49(30), 6411-9

The Actinomadura R39 DD-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small D-alanyl-D-alanine ... [more ▼]

The Actinomadura R39 DD-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small D-alanyl-D-alanine terminating peptides, especially those with a side chain that mimics the amino terminus of the stem peptide precursor to the bacterial cell wall. This paper describes the synthesis of (D-alpha-aminopimelylamino)-D-1-ethylboronic acid, designed to be a peptidoglycan-mimetic transition state analogue inhibitor of the R39 DD-peptidase. The boronate was found to be a potent inhibitor of the peptidase with a K(i) value of 32 +/- 6 nM. Since it binds some 30 times more strongly than the analogous peptide substrate, the boronate may well be a transition state analogue. A crystal structure of the inhibitory complex shows the boronate covalently bound to the nucleophilic active site Ser 49. The aminopimelyl side chain is bound into the site previously identified as specific for this moiety. One boronate oxygen is held in the oxyanion hole; the other, occupying the leaving group site of acylation or the nucleophile site of deacylation, appears to be hydrogen-bonded to the hydroxyl group of Ser 298. The Ser 49 oxygen appears to be hydrogen bonded to Lys 52. If it is assumed that this structure does resemble a high-energy tetrahedral intermediate in catalysis, it seems likely that Ser 298 participates as part of a proton transfer chain initiated by Lys 52 or Lys 410 as the primary proton donor/acceptor. The structure, therefore, supports a particular class of mechanism that employs this proton transfer device. [less ▲]

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