The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure–function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

Bacterial lytic transglycosylases (LTs) contribute to peptidoglycan cell wall metabolism and are potential drug targets to potentiate β‐lactam antibiotics to overcome antibiotic resistance. Since LT inhibitor development is underexplored, we probed 15 N‐acetyl‐containing heterocycles in a structure‐guided fashion for their ability to inhibit and bind to the Campylobacter jejuni LT Cj0843c. Ten GlcNAc analogs were synthesized with substitutions at the C1 position, with two having an additional modification at the C4 or C6 position. Most of the compounds showed weak inhibition of Cj0843c activity. Compounds with alterations at the C4 position, replacing the ‐OH with a ‐NH2, and C6 position, the addition of a ‐CH3, yielded improved inhibitory efficacy. All 10 GlcNAc analogs were crystallographically analyzed via soaking experiments using Cj0843c crystals and found to bind to the +1 +2 saccharide subsites with one of them additionally binding to the −2 −1 subsite region. We also probed other N‐acetyl‐containing heterocycles and found that sialidase inhibitors N‐acetyl‐2,3‐dehydro‐2‐deoxyneuraminic acid and siastatin B inhibited Cj0843c weakly and crystallographically bound to the −2 −1 subsites. Analogs of the former also showed inhibition and crystallographic binding and included zanamivir amine. This latter set of heterocycles positioned their N‐acetyl group in the −2 subsite with additional moieties interacting in the −1 subsite. Overall, these results could provide novel opportunities for LT inhibition via exploring different subsites and novel scaffolds. The results also increased our mechanistic understanding of Cj0843c regarding peptidoglycan GlcNAc subsite binding preferences and ligand‐dependent modulation of the protonation state of the catalytic E390.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell‐wall peptidoglycan (PG). Its non‐hydrolytic activity sustains cell‐wall remodeling and repair. We report herein our structure‐function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exo‐ and endolytic activities and forms the N‐acetyl‐1,6‐anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference of substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X‐ray crystallography and by MD simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di‐ and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.