PhD ThesisThe bacterial cell wall surrounds the cytoplasmic membrane and protects the cell
against osmolysis in addition to providing shape. The cell wall is comprised of
peptidoglycan, repeating units of N-acetly glucosamine and N-acetyl muramic acid
form glycan strands and are crosslinked by short peptides that contain both L- and
D-amino acids. Owing to the unique nature of peptidoglycan, and its absence in
eukaryotic organisms, the cell wall has become an important target for many
antibiotics, including the β-lactams and glycopeptides.
Newly synthesised peptidoglycan contains pentapeptides, which extend from the lactyl
moiety of the MurNAc sugar. These chains consist of L-alanine-D-γ-
glutamate/glutamine-L-lysine/meso-diaminopimelic acid-D-alanine-D-alanine. The
terminal D-alanine is often lost during cell wall maturation, either as a result of the
crosslinking reaction, in which the penultimate D-alanine is attached to the side-chain
of a neighbouring L-lysine or meso-diaminopimelic acid by an isopeptide bond, or as a
consequence of the activities of DD-carboxypeptidases, and results in a tetrapeptide.
The tetrapeptide can then be trimmed further to form a tripeptide by the action of
LD-carboxypeptidases. Although many DD-carboxypeptidases have been well
characterised, the majority of LD-carboxypeptidases that have been studied are active
only against peptidoglycan fragments and so cannot be responsible for producing the
tripeptides found in the cell wall. Of the LD-carboxypeptidases active against the
mature cell wall, DacB (Streptococcus pneumoniae), Csd6 (Helicobacter pylori) and
Pgp2 (Campylobacter jejuni), each has been shown to be essential in maintaining cell
morphology. It should be noted, however, that neither Csd6 nor Pgp2 share any
sequence similarity with DacB and belong to different peptidase families.
This thesis concerns the structural and biochemical characterisation of DacB, herein
renamed to LdcB (LD-carboxypeptidase B). The crystal structures of the apo form of
LdcB from both S. pneumoniae and Bacillus subtilis were solved, revealing a single
domain, globular protein with 2 sub-domains forming a V-shaped cleft in which the
active site is located. LdcB binds one zinc ion per monomer, located at the bottom of
the active site, and is a member of the LAS (lysostaphin, D-Ala-D-Ala peptidases, sonic
hedgehog) family of metalloproteins. Additionally, the activity of LdcB as an LDcarboxypeptidase
was confirmed and the crystal structure of LdcB from S. pneumoniae
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was solved in complex with a product mimic, M-Tri-Lys(D-Asn), revealing the molecular
basis for peptidoglycan recognition in this family of enzymes. Finally, the affinity of
LdcB for zinc and copper has been determined and it has been shown that catalysis is
not inhibited by the substitution of zinc by copper or cobalt.Sultanate of Oman Government, in particular
Sultan Qaboos University Hospital for providing the fundin
