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Review
. 2015 Oct 5;370(1679):20150031.
doi: 10.1098/rstb.2015.0031.

Activities and regulation of peptidoglycan synthases

Alexander J F Egan  1 , Jacob Biboy  1 , Inge van't Veer  2 , Eefjan Breukink  2 , Waldemar Vollmer  3
Affiliations
Review

Activities and regulation of peptidoglycan synthases

Alexander J F Egan et al. Philos Trans R Soc Lond B Biol Sci. .

Abstract

Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein-protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein-protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

Keywords: carboxypeptidase; glycosyltransferase; lipid II; penicillin-binding protein; peptidoglycan; transpeptidase.

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Figures

Figure 1.
Figure 1.
Reactions and domain organization of class A and class B PBPs. (a) Peptidoglycan synthesis and peptide cleavage reactions. A nascent glycan strand is synthesized from lipid II precursor by glycosyltransferase (GTase) reactions under the release of the undecaprenol pyrophosphate moiety (indicated by the zigzag line and two red dots). Peptide cross-links are formed by DD-transpeptidase (TPase) reactions catalysed by penicillin-binding proteins (PBPs), forming 4–3 cross-links. Some PBPs are also capable of hydrolysing the terminal d-alanine residue of the pentapeptide stem through DD-carboxypeptidase (CPase) activity, or hydrolysing the 4–3 cross-link through DD-endopeptidase (EPase) activity. (b) Crystal structures of E. coli PBP1B and PBP3. The bifunctional PBP1B (PDB ID: 3FWM) and the TPase PBP3 (PDB ID: 4BJP) anchor to the inner membrane (IM). The GTase domain of PBP1B is shown in blue, the TPase domains of both proteins are shown in green. The non-catalytic/regulatory domains such as the UB2H domain of PBP1B or the N-terminal module of PBP3 are shown in wheat. The residues essential for catalytic activity in each domain are labelled in red.
Figure 2.
Figure 2.
Synthesis of lipid II. (a) Schematic of the membrane-associated steps of lipid II synthesis. UDP-MurNAc-pentapeptide is attached to the lipid carrier undecaprenol phosphate by MraY creating lipid I. Next, UDP-GlcNAc is attached to lipid I by MurG creating lipid II, which can be modified at different positions as described in the text. (b) Undecaprenol can be extracted from leaves of Laurus nobilis using a mixture of acetone and hexane, followed by purification over a silica column and, if a uniform chain length is needed, by reversed-phase high-pressure liquid chromatography (HPLC). (c) UDP-MurNAc-pentapeptide can be extracted from Bacillus cereus (for the m-Dap version) or Staphylococcus simulans (l-lysine version) by blocking cell wall synthesis with vancomycin and subsequent boiling of the cells in water, centrifugation and lyophilization of the UDP-MurNAc-pentapeptide supernatant. (d) MraY and MurG are present in membranes from Micrococcus flavus, which are lysed and centrifuged to obtain the membrane vesicles in the supernatant. (e) To produce lipid II, all components are incubated for 2–4 h at room temperature, lipids are extracted using butanol/pyridine at pH 4.2, and lipid II is purified over a DEAE cellulose column.
Figure 3.
Figure 3.
Schemes of the currently used PG synthesis activity assays. (a) Separation of glycan strands by SDS–PAGE. (b) Continuous GTase assay with dansylated lipid II substrate. (c) Exchange reaction with radiolabelled d-Ala to monitor TPase activity. (d) In vitro GTase/TPase assay using lipid II substrate, with muramidase digestion and analysis of the resulting muropeptides by HPLC. Alternatively, the PG produced can be quantified by paper chromatography as shown in (c).
Figure 4.
Figure 4.
Class A PBPs exhibit carboxypeptidase activity at pH 5.0. Representative examples of HPLC chromatograms from PG synthesis assay (figure 3d) of PBP1B or PBP1A with radioactive lipid II with and without their cognate Lpo at either pH 7.5 or 5.0, as indicated. The resulting PG was digested with muramidase (cellosyl) yielding muropeptides, which were reduced with sodium borohydride and separated by HPLC. Radioactivity scale bars correspond to 500 cpm. Peak 1 corresponds to monophosphorylated disaccharide pentapeptide, peak 2 to disaccharide tetrapeptide, peak 3 to disaccharide pentapeptide, peak 4 to bis-disaccharide tetratetrapeptide, peak 5 to bis-disaccharide tetrapentapeptide and peak 6 to tris-disachharide tetratetrapentapeptide [107]. Peaks 2 and 4 are the result of DD-CPase activity, and are highlighted in red.
Figure 5.
Figure 5.
Activity of PBP1A and PBP1B in a membrane environment. (a) Incorporation of PBP1A and PBP1B into LUVs made of E. coli total lipid mixture. Samples were taken at various stages of proteoliposome preparation and resolved by SDS–PAGE followed by Coomassie blue staining. M, sample of the mixture of LUVs and purified protein; S, sample of the supernatant resulting from centrifugation of the mixture after detergent removal (Biobead treatment); P, LUVs with PBP. (b) Proteinase K digests PBP1A and PBP1B present in LUVs, suggesting that the PBPs were oriented outward. Untreated (–), proteoliposomes were pelleted by centrifugation without proteinase K treatment; treated (K), proteoliposomes were incubated with proteinase K prior to centrifugation; SDS extract (K/S), proteoliposomes were disrupted by SDS after proteinase K treatment to release any protein that was facing the interior of the LUV. All LUV-bound protein was accessible for proteinase K digestion, and no protein was detected in the interior of the LUVs. (c) Consumption of [14C]lipid II by PBP1A or PBP1B over time in LUVs. Amp, ampicillin; Moe, moenomycin.
Figure 6.
Figure 6.
Regulation of PG synthases in E. coli through protein–protein interactions. Scheme of the interactions of the major PG synthases of E. coli and their effects on enzyme activities and cellular localization. This figure is supplementary to table S1 which contains the references. An example of regulation in Vibrio cholerae which is not found in E. coli is also shown, distinguished by (Vibrio). Black lines: direct interaction. Blue arrow: recruitment to subcellular location, with the direction indicating the protein recruited. Green arrow: stimulatory effect with the direction indicating the affected synthase, the particular affected activity (GT and/or TP) is also indicated. Red arrow: negative modulation of PBP1B-LpoB TPase by CpoB, which is reversed by TolA as indicated by the capped black line. Grey-dashed arrow: uncharacterized role in regulation in the cell.
Figure 7.
Figure 7.
LpoB and FtsN synergistically enhance the GTase activity of PBP1B. (a) GTase activity of PBP1B was assayed by consumption of fluorescently labelled lipid II in vitro. Change in GTase rate is relative to PBP1B alone at the indicated reaction conditions and is shown as the mean ± s.d. (n = 4–12). Reaction conditions (Triton X-100 (TX-100) concentration, temperature and enzyme concentration) were optimized for each experiment. Specific conditions are indicated above the corresponding data. (b) A ternary complex of FtsN-PBP1B-LpoB was detected by in vitro cross-linking/pulldown approach. Proteins were cross-linked and applied to Ni-NTA beads. Cross-linkage of bound proteins was cleaved and samples separated by SDS–PAGE and visualized with Coomassie blue. FtsN-His retained LpoB only in the presence of PBP1B. The relatively weak retention of LpoB by PBP1B also occurs in the absence of FtsN, using His-PBP1B instead. This may be due to poor cross-linking efficiency between PBP1B and LpoB. A, applied sample; B, retained/bound protein.
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