Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa

Abstract

β-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. , a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.

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

“…Notably, it is only after the binding of substrates that contain pentapeptide stems that it can exhibit endolytic activity due to a conformational change of the protein on substrate binding. A movie of this rearrangement is available in the supplementary material of reference [23]. Additional studies suggest protein-protein interactions with inner membrane PBPs are also important [26].…”

“…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].…”

“…X-ray crystal structures of Slt in its apo form as well as in complex with various synthetic PG substrates and reaction products demonstrated that this Lt has both exolytic and endolytic activity [23]. It is a donut shaped protein like Slt of E. coli.…”

Constructing and deconstructing the bacterial cell wall

“…283,284 Accordingly, we have undertaken extensive mechanistic and structural studies of this family. These efforts have led us to the discovery that an ABC-transporter-like regulatory module is present in the structure of the P. aeruginosa MltF LT 285 ; that the P. aeruginosa SltB1 LT has a catenane dimer structure; that the P. aeruginosa Slt LT 286 has the same compelling doughnut-shape as is seen in other Slt LTs whose catalytic activity is governed by important conformational changes 234,[287][288][289] ; and to a computational mechanism for the smallest LT of E. coli, MltE, 233 based on our crystallographic analyses. 290,291 The assertion of an intimate relationship between the LT family and β-lactam antibiotic efficacy has irrefutable support.…”

“…303 F I G U R E 3 The stereo image of the structure of the complex of the E503Q catalytic-impaired mutant of the P. aeruginosa Slt LT bound to bulgecin A (bottom structure) (PDB 6FCQ). 286 Bulgecin A (depicted in sphere representation; color by atom types) is bound at the active site of this LT. This active site is located on the threshold of the central opening of this doughnutshaped protein…”