The direction of glycan chain elongation by peptidoglycan glycosyltransferases - PubMed (original) (raw)

. 2007 Oct 24;129(42):12674-5.

doi: 10.1021/ja075965y. Epub 2007 Oct 3.

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The direction of glycan chain elongation by peptidoglycan glycosyltransferases

Deborah L Perlstein et al. J Am Chem Soc. 2007.

Abstract

Peptidoglycan glycosyltransferases (PGTs) are highly conserved enzymes that catalyze the polymerization of Lipid II to form the glycan strands of bacterial murein. Because they play a key role in bacterial cell wall synthesis, these enzymes are potentially important antibiotic targets; however, their mechanisms are not yet understood. One longstanding question about these enzymes is whether they elongate glycan chains by adding subunits to the anomeric (reducing) end or to the 4-hydroxyl (non-reducing) end. We have developed an approach to test the direction of chain elongation that involves the use of nascent peptidoglycan chains which are blocked at their non-reducing ends. In the presence of the PGT domains of Staphylococcus aureus PBP2, Aquifex aeolicus PBP1A, Escherichia coli PBP1A or Escherichia coli PBP1B, these blocked substrates react with Lipid II to form longer glycan chains. These results establish that PGTs elongate nascent peptidoglycan chains by the addition of disaccharide subunits to the anomeric (reducing) end of the growing polymer.

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Figures

Figure 1

Figure 1

Natural Lipid II as well Lipid II (1), Lipid IV (3), and Lipid VIII (5) substrate analogs and their Gal-labeled versions (2, 4, and 6).

Figure 2

Figure 2

Models for the direction of glycan polymerization. A) Scheme showing that a glycan strand can be extended by adding new units to the non-reducing end (left) or to the reducing end (right). B) Scheme showing our strategy to test the direction of chain elongation. If the PGT is incubated with Lipid II and a polymer substrate blocked at the non-reducing end with [14C]Gal, then the polymer will only be extended if new units add to the reducing end.

Figure 3

Figure 3

Gel electrophoresis assays to determine the direction of elongation. A) Scheme for the synthesis of the Gal-oligomer substrates. B) Gel electrophoresis of PGT reaction mixtures. Each lane contains either [14C]-Gal-Lipid IV (Lane 1) or the [14C]-Gal-oligomer mix (Lanes 2-9) incubated in the presence or absence of PGT and Lipid II. The PGTs investigated include E. coli PBP1A (Lanes 3-4), E. coli PBP1B (Lane 5), A. aeolicus PBP1A (Lane 6-7), and S. aureus PBP2 (Lane 8-9). The identities of the Gal-labeled oligomers, as assessed by electrophoretic mobilities (Figure S2) and MS analysis of the unlabeled reaction mixture (Table S1), are indicated. Asterisked products arise late in the reaction, presumably as a result of hydrolysis or transglycosylation with diphospholipid. They do not affect the analysis.

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