Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus

Abstract

The peptidoglycan of Staphylococcus aureus is highly amidated. Amidation of α-D-isoglutamic acid in position 2 of the stem peptide plays a decisive role in the polymerization of cell wall building blocks. S. aureus mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin, indicating that targeting the amidation reaction could be a useful strategy to combat this pathogen. The enzyme complex that catalyzes the formation of α-D-isoglutamine in the Lipid II stem peptide was identified recently and shown to consist of two subunits, the glutamine amidotransferase-like protein GatD and the Mur ligase homolog MurT. We have solved the crystal structure of the GatD/MurT complex at high resolution, revealing an open, boomerang-shaped conformation in which GatD is docked onto one end of MurT. Putative active site residues cluster at the interface between GatD and MurT and are contributed by both proteins, thus explaining the requirement for the assembled complex to carry out the reaction. Site-directed mutagenesis experiments confirm the validity of the observed interactions. Small-angle X-ray scattering data show that the complex has a similar conformation in solution, although some movement at domain interfaces can occur, allowing the two proteins to approach each other during catalysis. Several other Gram-positive pathogens, including Streptococcus pneumoniae, Clostridium perfringens and Mycobacterium tuberculosis have homologous enzyme complexes. Combined with established biochemical assays, the structure of the GatD/MurT complex provides a solid basis for inhibitor screening in S. aureus and other pathogens.

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

“…Using two complementary approaches, the in vitro pulldown assay and the in vivo interaction assay, we established that DUF1727 is responsible for the interaction between the two proteins MurT and GatD and for complex formation. This observation was confirmed by recent crystallographic structures, where GatD docks to the C terminus of MurT (20,21). Our results, obtained using distinct approaches, showed that DUF1727 is sufficient for the interaction with GatD and, more importantly, is essential for complex activity.…”

“…The recently published structures of the MurT-GatD complexes from S. pneumoniae and S. aureus revealed a unique role for the C-terminal domain of MurT (DUF1727). Residue D349 plays a pivotal role in glutaminase activity by completing the catalytic triad (21). Our MurT-D349A mutation was responsible for a loss of complex formation and was not able to complement the amidation phenotype, which we attribute to the loss of protein stability and, consequently, its function (Fig.…”

“…Combined, these results show that this residue has a pivotal role in the catalytic mechanism of the complex. The active site of GatD includes C94, H128, and MurT-D349 (21,25), and it has been shown that residues Y17 and R128 of GatD are important in blocking the substrate path to the active site, having a regulatory role in glutaminase activity (25). In the MurT-GatD structure from S. aureus, the GatD-Y17 residue can adopt two distinct conformations.…”

Cell wall peptidoglycan inMycobacterium tuberculosis: An Achilles’ heel for the TB-causing pathogen

“…2018; Noldeke et al . 2018). While GatD is inactive in the absence of MurT, the latter can functionally substitute the role of MurE (Munch et al .…”

Homology Modelling, Phylogenetic Analysis, and Molecular Docking of Glutamine Aminotransferase, gatD from Clostridium botulinum

“…The peptide moiety in the lipid II undergoes secondary modifications prior to the polymerization reactions such as amidation, the addition of extra amino acids, and esterification, etc. Recently, the crystal structure of an enzyme, GatD/MurT complex from S. aureus was solved at high resolution [2]. Amidation of the α-carboxyl group of the D-isoglutamate residue in Lipid II results in the formation of D-isoglutamine [3].…”