The mechanism behind the selection of two different cleavage sites in NAG-NAM polymers

Abstract

Peptidoglycan is a giant molecule that forms the cell wall that surrounds bacterial cells. It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues connected by -(1,4)-glycosidic bonds and cross-linked with short polypeptide chains. Owing to the increasing antibiotic resistance against drugs targeting peptidoglycan synthesis, studies of enzymes involved in the degradation of peptidoglycan, such as N-acetylglucosaminidases, may expose new, valuable drug targets. The scientific challenge addressed here is how lysozymes, muramidases which are likely to be the most studied enzymes ever, and bacterial N-acetylglucosaminidases discriminate between two glycosidic bonds that are different in sequence yet chemically equivalent in the same NAG-NAM polymers. In spite of more than fifty years of structural studies of lysozyme, it is still not known how the enzyme selects the bond to be cleaved. Using macromolecular crystallography, chemical synthesis and molecular modelling, this study explains how these two groups of enzymes based on an equivalent structural core exhibit a difference in selectivity. The crystal structures of Staphylococcus aureus N-acetylglucosaminidase autolysin E (AtlE) alone and in complex with fragments of peptidoglycan revealed that N-acetylglucosaminidases and muramidases approach the substrate at alternate glycosidic bond positions from opposite sides. The recognition pocket for NAM residues in the active site of N-acetylglucosaminidases may make them a suitable drug target.

Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

“…The positioning of the conserved core helices is equivalent to that of AtlE in seven other known GH73 crystal structures 16 , the only two structures that substantially deviate are SagB and Atl-gl ( Supplementary Fig. 1).…”

“…The superimposition parameters slightly deviate from a proper noncrystallographic symmetry dimer. The structure of SagB shares the topological layout and heart-like shape of AtlE (4PIA) 16 . it is composed of right (R) and left (L) domains divided into core and lobe regions (Fig.…”

“…Their substrate is a component of the bacterial cell wall, a carbohydrate polymer composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). We recently determined the crystal structures of a homologous protein, autolysin E (AtlE) in complex with substrate fragments, which we used together with other available structural data on lysozymes to build a model of (NAG-NAM) 3 substrate binding to AtlE 16 . We have also analyzed the amino acids that are important for the reaction to transpire and identified E138 as the catalytic general acid and Y224-A225-S226-D227 as the motif responsible for substrate binding 17 .…”

“…AtlA has been shown to be localized at the septal region and to be involved in cell division 14,21 . We combined the understanding of substrate binding obtained from our previous AtlE studies 16,17 with insights obtained from the SagB and AtlA-gl structures. The analysis of substrate binding sites revealed that for the active site residues to enable the enzymatic reaction to proceed, a substantial conformational change has to take place.…”

Structure and reconstitution of a hydrolase complex that releases peptidoglycan from the membrane after polymerization

“…A groove extends from this opening along the extracellular face of SpdC all the way to the active site pocket of SagB. Several lines of evidence have established the directionality of oligosaccharide binding for the family of glucosaminidases to which SagB belongs 28 , and this directionality is consistent with nascent peptidoglycan following the groove such that the non-reducing end of the polymer exits toward the cell wall (Supplementary Fig. 22).…”