First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD

Abstract

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

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

“…1 ). As previously postulated 19 and recently shown 24 , GatD exhibits a class-I glutamine amidotransferase-like fold. A DALI search 25 identifies the enzymes HisH, PdxT and PurQ from Thermotoga maritima as the closest structural homologs (Z-values of 17.4, 15.8 and 15.0, respectively).…”

Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae

“…The corresponding residues in S. aureus GatD are Cys94-His189-Glu19. Glycine substitution of Cys94 in S. aureus GatD abolished amidation of lipid II 4 , and substitution of either Cys94 or His189 by alanine suppressed a residual glutaminase activity in the absence of MurT 38 . The glutamate of the triad in canonical class I glutaminases is highly conserved, two residues downstream of the catalytic histidine in sequence, but its catalytic role is uncertain as its replacement with a non-acidic residue had minor effect on the glutaminase activity of the human γ-glutamyl hydrolase or the Escherichia coli carbamoyl phosphate synthase 39 , 40 .…”

“…Conserved GD Arg142 at the extremity of the catalytic cleft opposite GD Cys107 appears to interact with the glutamine substrate. The corresponding Arg128 in S. aureus GatD was found to interact with a glutamine outside of the active site, and its replacement by an alanine abrogated glutaminase activity 38 . The two structures support a model in which the side chain of GD Arg142 helps to “capture” the substrate from the solution (Fig.…”

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

“…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.…”