Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus

Abstract

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum

“…Recently, it was shown that the enzyme LtsA is responsible for the amidation of mDAP (46). The amidation of D-Glu is probably performed by Cg0299 and Cg0300, as shown for homologous proteins of Staphylococcus aureus (47,48). Subsequently, Lipid II is translocated across the cell membrane (step 2), where the peptidoglycan polymerization takes place.…”

Hydrolysis of peptidoglycan is modulated by amidation of meso‐diaminopimelic acid and Mg2+ in Bacillus subtilis

“…In Staphylococcus aureus and Streptococcus pneumoniae where the di‐basic amino acid is L‐Lys instead of mDAP, D‐Glu is amidated to D‐iso‐glutamine. The two enzymes responsible for D‐Glu amidation (the MurT/GatD complex) have been identified (Munch et al ., 2012; Figueiredo et al ., 2012), and PG transpeptidases have been shown to work more efficiently when D‐Glu is amidated (Munch et al ., 2012; Figueiredo et al ., 2012). In B. subtilis , mDAP was found to be amidated in most muropeptides of vegetative cell PG, while D‐Glu was not amidated (Atrih et al ., 1999; Warth and Strominger, 1971).…”

Structural Variations of the Cell Wall Precursor Lipid II and Their Influence on Binding and Activity of the Lipoglycopeptide Antibiotic Oritavancin

“…Large-scale synthesis and purification of the peptidoglycan precursors lipid I, lipid II, lipid II-Gly 1Ϫ5 and amidated lipid II were performed as described previously (45,46). Lipid II-Asp was synthesized with purified lipid II and purified recombinant Asl-His 6 as described above using nonradioactive D-aspartate (37,38).…”