Turnover of Bacterial Cell Wall by SltB3, a Multidomain Lytic Transglycosylase of Pseudomonas aeruginosa

Abstract

A family of 11 lytic transglycosylases in Pseudomonas aeruginosa, an opportunistic human pathogen, turn over the polymeric bacterial cell wall in the course of its recycling, repair and maturation. The functions of these enzymes are not fully understood. We disclose herein that SltB3 of P. aeruginosa is an exolytic lytic transglycosylase. We characterize its reaction and its products by the use of peptidoglycan-based molecules. The enzyme recognizes a minimum of four sugars in its substrate, but can process a substrate comprised of a peptidoglycan of 20 sugars. The ultimate product of the reaction is N-acetylglucosamine-1,6-anhydro-N-acetylmuramic acid. The X-ray structure of this enzyme is reported for the first time. The enzyme is comprised of four domains, arranged within an annular conformation. The polymeric linear peptidoglycan substrate threads through the opening of the annulus, as it experiences turnover. Graphical AbstractCell wall, a crosslinked polymer that encases the entire bacterium, is critical for survival. The peptidoglycan, the major constituent of cell wall, is comprised of a linear polymer of repeating N-acetylglucosamine (NAG)-N-acetylmuramic acid (NAM) disaccharide, with a Corresponding Authors: mobashery@nd.edu and xjuan@iqfr.csic.es. 1 The first two authors contributed equally Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Tables S1-S2, Figures S1-S7 (PDF). The crystallographic coordinates are deposited in the Protein Data Bank (PDB codes 5anz, 5ao7 and 5ao8). (Figure 1). Crosslinking of the neighboring peptidoglycan strands takes place at the peptide stems. The mature cell wall is a single molecule of significant complexity, which is assembled and recycled in a dynamic manner. 1 Recycling of cell wall takes place in response to damage (due to antibiotics, for example), to construction of complex macromolecular assemblies such as the flagellum, or to processing during replication that requires changes to the cell wall. 2,3 The breakdown of the peptidoglycan for the recycling events is performed by lytic transglycosylases (LTs), enzymes that exist within the periplasmic space of Gram-negative bacteria in either soluble or membrane-bound forms. These enzymes perform important biological functions, which are the subjects of intense study. The importance of their functions is underscored by their multiplicity-Escherichia coli has eight 3-5 and Pseudomonas aeruginosa has eleven 6 -and by redundancies of the reactions that they perform. For example, six of the enzymes of E. coli could be ablated without any apparent consequence, but the organism cannot survive the loss of seven. 4 The mechanism of reaction of LT has been studied previously. 3,7,8 These enzymes bind to the peptidoglycan and degrade it either by removal of a disaccharide from the ends of the linear polymer (the exolytic reaction) or by cleavage of the polymer in the middle (the endolytic reaction). Whereas the catalytic sites of LTs bear resemblance to the...

The hydrolysis mechanism of a GH45 cellulase and its potential relation to lytic transglycosylase and expansin function

“…Although experimental characterization of the HiCel45A D10N mutant revealed no hydrolytic activity (5), this does not preclude other catalytic activity in the HiCel45A D10N mutant, similar to MltA or expansins. The role of aspartic/glutamic acid as the sole catalytic residue mediating glycosidic bond lysis is the established mechanism in LTs (1,11,(15)(16)(17)32,33). Figure 5A illustrates the mechanism proposed in the HiCel45A D10N mutant, which involves the initiation of glycolysis by proton transfer from the acid (D121) to the glycosidic oxygen, followed by the transfer of the hydroxymethyl hydroxyl proton to D121 acting as a base.…”

“…Expansins facilitate growth in plant cell walls (10,12), but hydrolytic products of their action have not been detected (13). LTs non-hydrolytically cleave glycosidic linkages in bacterial cell wall peptidoglycans (14) thus forming an anhydro glycan product (15)(16)(17). Expansins as well as one family of LTs (called family 2 in the classification scheme of Blackburn and Clarke (18), classified as GH102 in CAZy, and represented by Escherichia coli MltA (11)) share a similar double-psi β-barrel fold with GH45s and are structurally similar at the active site.…”

Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) Pseudomonas aeruginosa

“…Recently, lytic transglycosylases of P. aeruginosa have been extensively characterized [21][22][23][24][25][26][27]. These cell wall proteins are found in many other pathogenic bacteria and are classified according to amino acid sequence and function [28].…”

“…SltB1 [22] and SltB3 [24] have also been studied using x-ray crystallography. SLtB1 protein structures suggest that the protein forms a so-called "catenane" homodimeric structure in which the active sites face one another and are thus completely occluded.…”

“…It is speculated that this soluble dimer may represent a form of activity regulation [22]. SltB3 is an exolytic enzyme with four distinct enzymatic domains within the donut shaped annular protein [24]. SltB3 can recognize PG substrates that are 4-20 sugars in length.…”