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

Abstract

To achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

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

“…Consistent with previous findings, SagB lacking its TM helix has low activity ( Fig. 2b , lane 6; also Extended Data 1 ) 13 , 20 . In contrast, full-length SagB fully converted the peptidoglycan oligomers to diffuse bands high in the gel ( Fig.…”

“… 24 , 25 LC-MS analysis confirmed the presence of a diphosphate on a peptidoglycan fragment having an odd number of sugars, consistent with glucosaminidase cleavage to leave a terminal MurNAc ( Supplementary Figure 8 ). Notably, SagB-SpdC’s cleavage activity in vitro depended on the conserved catalytic glutamate 13 , 26 13 , 26 13 , 13 , 25 13 , 25 , 20 in SagB (E155), but not on an SpdC residue conserved in eukaryotic CAAX proteases and required for their proteolytic activity ( Supplementary Figure 9 ). Moreover, cellular phenotypes of the SagB catalytic mutant resemble a sagB knockout, whereas SpdC mutants lacking putative catalytic residues resemble wild-type ( Extended Data 3 ) 15 .…”

Biochemical and Structural Characterization of Two Cif-Like Epoxide Hydrolases fromBurkholderia cenocepacia

“…Structural flexibility is a critical factor in enzymatic catalysis. Flexibility plays an important role in substrate specificity and turnover, and it is perhaps especially important for enzymes in which two or more domains contribute to the active site architecture [29,30]. Indeed, protein scientists have learned from nature to leverage this principle and manipulate enzyme dynamics to create new functionalities [31].…”

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

“…2b, lane 6; also Supplementary Fig. 5) 13,20 . In contrast, full-length SagB fully converted the peptidoglycan oligomers to diffuse bands high in the gel (Fig.…”

“…10). Notably, SagB-SpdC’s cleavage activity in vitro depended on the conserved catalytic glutamate 13,26 13,26 13,25 13,25,20 in SagB (E155), but not on an SpdC residue conserved in eukaryotic CAAX proteases and required for their proteolytic activity (Supplementary Fig. 11).…”