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. 2014 Oct 22;136(42):14678-81.
doi: 10.1021/ja508147s. Epub 2014 Oct 10.

Detection of lipid-linked peptidoglycan precursors by exploiting an unexpected transpeptidase reaction

Yuan Qiao  1 , Matthew D Lebar, Kathrin Schirner, Kaitlin Schaefer, Hirokazu Tsukamoto, Daniel Kahne, Suzanne Walker
Affiliations

Detection of lipid-linked peptidoglycan precursors by exploiting an unexpected transpeptidase reaction

Yuan Qiao et al. J Am Chem Soc. .

Abstract

Penicillin-binding proteins (PBPs) are involved in the synthesis and remodeling of bacterial peptidoglycan (PG). Staphylococcus aureus expresses four PBPs. Genetic studies in S. aureus have implicated PBP4 in the formation of highly cross-linked PG, but biochemical studies have not reached a consensus on its primary enzymatic activity. Using synthetic Lipid II, we show here that PBP4 preferentially acts as a transpeptidase (TP) in vitro. Moreover, it is the PBP primarily responsible for incorporating exogenous d-amino acids into cellular PG, implying that it also has TP activity in vivo. Notably, PBP4 efficiently exchanges d-amino acids not only into PG polymers but also into the PG monomers Lipid I and Lipid II. This is the first demonstration that any TP domain of a PBP can activate the PG monomer building blocks. Exploiting the promiscuous TP activity of PBP4, we developed a simple, highly sensitive assay to detect cellular pools of lipid-linked PG precursors, which are of notoriously low abundance. This method, which addresses a longstanding problem, is useful for assessing how genetic and pharmacological perturbations affect precursor levels, and may facilitate studies to elucidate antibiotic mechanism of action.

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Figures

Figure 1
Figure 1
Schematic depicting carboxypeptidase (CP) and transpeptidase (TP) activities of penicillin-binding proteins that make peptidoglycan (a), and structures of the lipid-linked building blocks used (b).
Figure 2
Figure 2
S. aureus PBP4 has TP activity in vitro. (a) Schematic of assay to monitor PBP4 activity. The PBP4-activated substrate adduct can be attacked by water or X. Three possible degradation products are yielded: A represents unreacted muropeptide, B is the hydrolysis product, and C is a TP product with Gly2 or d-Ser incorporated. (b) LC/MS extracted ion chromatograms (EICs) of a control reaction without PBP4 (i), a reaction with PBP4 (ii), and reactions containing PBP4 and Gly2 (iii) or d-Ser (iv). (M+2H)/2 ions: A, 485.2; B, 449.6; C(Gly2), 506.7; C(d-Ser), 493.2. See Figure S2a for other Glyx traces.
Figure 3
Figure 3
S. aureus PBP4 has TP activity in vivo. (a) Structures of functionalized d-lysine probes (FDL and BDL). (b) Mid-log phase S. aureus cells (wildtype and Δpbp4) were treated with FDL (4 μM) for 10 min. Images were adjusted to the same intensity scale for comparison. Scale bar, 2 μm.
Figure 4
Figure 4
S. aureus PBP4 has promiscuous TP activity. (a) Schematic for analyzing PBP4 substrate tolerance. (b) LC/MS EICs of PBP4 reactions with preformed PG (i) and Lipid II (ii) both show the d-Tyr-containing muropeptide product peak C. (M+2H)/2 ions were extracted: A, 485.2; B, 449.6; C, 531.2.
Figure 5
Figure 5
S. aureus PBP4 enables detection of cellular lipid-linked PG precursors. (a) Schematic of the chemoenzymatic route to BDL-Lipid I and Lipid II analogues. (b) Western blot shows the change in cellular levels of PG precursors upon 10 min of antibiotic treatments. Lipid-linked PG precursors were extracted from 2 mL of S. aureus culture, subjected to BDL labeling, and blotted with streptavidin-HRP.
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