Abstract:
The bacterial cell wall with its main structural component, peptidoglycan, is essential for the
survival of bacteria as it maintains cell integrity by countering the internal turgor pressure and
provides protection against environmental stresses as an outer defense barrier. Continuous
synthesis, remodeling, and degradation of the peptidoglycan macromolecule is required during
the bacteria's life cycle in order to allow growth, division, and separation. In the context of
increasing antibiotic resistance in many bacterial strains, some of which are human pathogens
with major implications for clinical healthcare, these processes and the peptidoglycan molecule
itself have been the subject of numerous studies.
In the course of peptidoglycan biosynthesis, many bacteria introduce modifications to the
archetypical peptidoglycan structure to evade host defense mechanisms and to improve rigidity
and stability of the peptidoglycan scaffold. One of the most common modifications is the
amidation of the second amino acid residue in the peptidoglycan stem peptide, which directly
influences the peptidoglycan cross-linking efficiency and contributes to antibiotic resistance. In
the first project of this thesis, the MurT/GatD enzyme complex that performs this amidation in
important human pathogenic bacteria such as Staphylococcus aureus, Mycobacterium
tuberculosis, and Streptococcus pneumoniae was investigated. Following up on two previously
determined structures, a new crystal packing for S. aureus MurT/GatD was identified that
allows crystallization experiments with peptidoglycan precursor ligands. Moreover, a third
homologous structure from Streptococcus pyogenes was obtained enabling a more detailed
comparison of two distinct arrangements of the protein complex and confirming the conserved
binding site for ATP MurT in a ligand-bound structure. The structures determined in this work
in combination with enzyme activity and ligand binding assays expanded and improved our
knowledge on bacterial peptidoglycan amidation by the MurT/GatD complex. Together with
the structures from S. aureus and S. pneumoniae, they provide a basis for future structural
studies on MurT/GatD with the long-term goal of developing specific, small molecule inhibitors
for this enzyme complex in order to combat antimicrobial resistant pathogens.
To preserve resources when degrading the cell wall, most bacteria recycle the individual
building blocks of the peptidoglycan molecule, either for reuse in cell wall synthesis or in case
of starvation as a nutrient source. Recycling pathways influence the viability of bacterial species
and are often linked to antibiotic resistance mechanisms. While peptidoglycan recycling in
Gram-negative bacteria has been studied in more detail, less information is available for Gram-
Ipositive species. The second project of this thesis focused on two peptidases, YkfA and YkfC,
from the Gram-positive model organism Bacillus subtilis that cleave amide bonds in
peptidoglycan-derived peptides. Protein expression, purification, and crystallization
experiments were conducted for the L,D-carboxypeptidase YkfA, that cleaves the bond
between the third and fourth residue in the peptidoglycan stem peptide, with the aim of
determining the first atomic structure of this enzyme. The experiments provide a starting point
for further studies to elucidate the structure and mechanism of YkfA in order to generally
improve the understanding of serine peptidases from the S66 family that are involved in cell
wall recycling. The D,L-endopeptidase YkfC cleaves between the second and third residue in
the stem peptide and belongs to the major class of NlpC/P60 cysteine peptidases. In this work,
a high yield expression and purification protocol of YkfC was established followed by X-ray
crystallographic experiments. To analyze substrate specificity and engagement on an atomic
level and for comparison to a homologous structure from Bacillus cereus, structures of YkfC
in the apo state, covalently bound to a dipeptide product, and in complex with a pentapeptide
substrate were determined. Investigation of the structures revealed a conserved specificity to
peptides with a free N-terminal L-alanine residue and the formation of an oxyanion hole by a
tyrosine residue, which is unusual for cysteine peptidases, in the YkfC subfamily. Moreover,
the structures gave insights into the orientation of important residues during catalysis and the
engagement of the C-terminal residues in the peptide substrate by YkfC. Residues potentially
forming contacts to the peptide substrate were additionally investigated in a mutation activity
experiment. With the here laid structural foundation, further experiments including the natural
mDAP-containing peptide substrate are now straightforward. Afterwards, the focus could be
shifted towards related NlpC/P60 peptidases that, in contrast to B. subtilis YkfC, are essential
for the bacterium.
In summary, the structural studies on peptidoglycan modification and recycling enzymes
presented in this work improved our understanding of important processes associated with the
bacterial cell wall. The information gathered could serve as a basis for comparison in future
studies on functionally related enzymes and help in the development of strategies to inhibit
these enzymes and thus combat bacterial pathogens.
Peptidoglycan