Structure of the cell wall PG in S. aureus. (A) Schematic overview of the cell wall in S. aureus. (B) Structure of the peptidoglycan. (C) Schematic presentation of peptides used to study target bond specificity of the enzymes. The panel A is adapted from “Gram-Positive Bacteria Cell Wall Structure”, by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Workflow to study M23 PGH substrate specificities. Panels in the upper left corner, the two main strategies used in the study. Kinetic measurements carried out with PG fragments (synthetic peptides) were supported by bacteria-based kinetic measurements using S. aureus USA300 cells. Panels in the upper right corner, (A) hydrolysis of synthetic pGly (mM) by LSS (green) and LytM (magenta) monitored by 1H NMR spectroscopy over time (h). Both enzymes were used at the concentration of 50 μM. (B) Rate of hydrolysis (mM/min) of pGly derived from A in the first 60 min of the reaction for LSS and LytM. (C) 13C-HMBC NMR spectrum showing the end-point kinetic of LSS-treated muropeptides extracted from S. aureus USA300 cells. (D) Turbidity assay using S. aureus USA300 cells in the presence of LSS and LytM at a concentration of 3 μM. The cell lysis is expressed as percentage reduction of the bacteria suspension optical density at 600 nm over time (h). (E) Pentaglycine hydrolysis by LSS and LytM was studied by using a Gly2 13C-labelled substrate. (F) NMR pulse sequence for the acquisition of glycine Hα-detection optimised 2D HA(CA)CO spectra, showing correlations between 1Hα and 13CO atoms. (G) With a label the otherwise identical products of the two hydrolysis reactions G2-G3, G3-G4 can now be differentiated. (H) The order of appearance of the peaks of the labelled products as a function of time. The labelled glycine has a different 13CO shift when as G2 in triglycine or G2 in diglycine. (I) Heatmap summarising the bond preferences for the enzymes in the pGly. Hydrolysis of peptides 1-7 by LSS (green) and LytM (magenta). (J) Initial rates of substrate hydrolysis (mM/min) of LSS and LytM at 2 μM concentration and (K) the same rates normalised to that of pGly. (L) Absolute values of rates of hydrolysis. For PG fragments 2 and 3, two independent measurements were performed to test and accredit the reproducibility of the method (see Materials and Methods).

Representative examples of real time NMR monitoring of substrate hydrolysis. Quantitative 1H spectra at selected time points in the hydrolysis reactions of peptide 2 by LSS (A) and LytM (B). In hydrolysis by LSS peaks of Ala Hα in products KDAG and KDAGG gradually appear as a function of time, whereas in LytM reaction KDA and KDAG are formed. Time points are given in minutes next to the spectra. Peak assignments in the reference spectrum are the following: 1 Ala Hα, 2 Gly1-Gly4 Hα, 3 Lys Hα, 4 Gly5 Hα, 5 Lys Hε, 6 Lys Hβ, 7 Lys Hδ, 8 Lys Hγ, 9 Ala Hβ. Asterisk marks the peak of the buffer. The alanine Hα quartets of substrate (4.373 ppm) and KDAGG (4.365 ppm) partially overlap. (C, D) On the left, concentrations in function of reaction time derived from NMR peak integrals from a typical reaction setup with 0.4 mM peptide and 2 μM LSS or 50 μM LytM. On the right, relative product concentrations at reaction end points for the studied PG fragments. (E, F) Rates of formations of products in hydrolyses by LSS and LytM of the studied PG fragments 1-7. (G) Bonds cleaved by LSS and LytM in different PG fragments.

Hydrolysis of PG fragments with a shorter cross-bridge or with serine in cross-bridge. Rates of substrate hydrolysis of fragments 8 and 9 as compared with 7 by LSS (green) and LytM (magenta) (A) and formation of product(s) in hydrolysis by LSS (B) and LytM (C). Rates of substrate hydrolysis of fragments 10 and 11 as compared with 2 (D) and formation of product(s) in hydrolysis by LSS (E) and LytM (F). Depictions of structures of used PG fragments (G). LytM was used at a concentration of 50 μM while LSS was 2 μM.

Substrate specificity of LSS and LytM.

Schechter and Berger nomenclature is employed to describe the differences in substrate specificity between LSS (A) and LytM (B). Scissile bond in the substrate is between the P1 and P1’ positions, indicated by green (LSS) and purple arrows (LytM), and hence residues towards the N-terminus from the scissile bond are P1-P4, whereas those towards the C-terminus are designated as P1’-P4’. PG fragments devoid of stem peptide linked to the C-terminal glycine are shown aligned with respect to their cleavage sites together with the rate of hydrolysis of the particular scissile bond. Consensus sequence displays preferable amino acid(s) that are accepted in the specific position (…P2, P1, P1’, P2’…) with respect to the cleavage site. Red circles/ovals indicate missing or less than optimal amino acid accommodation in the particular P site, which translates into reduced catalytic efficiency. Serine substitutions in the glycine bridge and associated rates of hydrolysis are indicated by red and orange colors. (C-F) show the docking results for fragment 2 into the catalytic site of LSS and LytM. (G) shows docking result of fragment 11 to LSS. (H-J) show the docking results for fragments 10 and 11 into the catalytic site of LytM. LSS and LytM are capable of cleaving the Gly1-Gly2 bond in 2 (C, D). LytM is also able to cleave the D-Ala-Gly1 bond (F), however, in LSS this would result in a steric clash between the D-Ala side chain and the residues in loop 1 (E). LytM was used at a concentration of 50 μM while LSS was 2 μM.