Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism

Abstract

Bacterial transpeptidase and transglycosylase on the surface are essential for cell wall synthesis, and many antibiotics have been developed to target the transpeptidase; however, the problem of antibiotic resistance has arisen and caused a major threat in bacterial infection. The transglycosylase has been considered to be another excellent target, but no antibiotics have been developed to target this enzyme. Here, we determined the crystal structure of the Staphylococcus aureus membrane-bound transglycosylase, monofunctional glycosyltransferase, in complex with a lipid II analog to 2.3 Å resolution. Our results showed that the lipid II-contacting residues are not only conserved in WT and drug-resistant bacteria but also significant in enzymatic activity. Mechanistically, we proposed that K140 and R148 in the donor site, instead of the previously proposed E156, are used to stabilize the pyrophosphate-leaving group of lipid II, and E100 in the acceptor site acts as general base for the 4-OH of GlcNAc to facilitate the transglycosylation reaction. This mechanism, further supported by mutagenesis study and the structure of monofunctional glycosyltransferase in complex with moenomycin in the donor site, provides a direction for antibacterial drugs design.

Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase

“…All the MtgA variants used are without transmembrane (TM) segment, this deletion does not affect the structure of the enzyme [18]. Deletion of the TM region was found to reduce the activity of the enzyme, most probably because TM contributes to the hydrophobic interaction with the substrate [19]. This construct is more suited for our studies because it will better highlight the specificities of the enzyme for the polar regions (sugars, peptide and phosphates) of substrate analogs.…”

“…in the apo form and in complex with moenomycin A and a lipid II analog [15][16][17][18][19][20]. These structures show that the GT is composed of a lysozyme-like globular domain and a small hydrophobic domain (called jaw domain) with a deep cleft between them containing the active site (Fig.…”

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

“…In the free inner membrane-embedded enzyme, to start the catalytic cycle, lipid II binds to the donor site, and a second lipid II molecule binds the extended ␣4 helix loop at the acceptor site, which is observed unperturbed by crystal packing for the first time in our E. coli PBP1b-aztreonam crystal structure (Fig. 6, A-D) (6,26,29). Upon formation of the precatalytic complex with both donor-and acceptor-bound, the general base Glu 233 , which we now observe directly in the electron density of the PBP1b GTase active site (Figs.…”

“…In one perspective, the negative charge on pyrophosphate is stabilized by the universally conserved Glu 290 either directly or via an intervening magnesium ion (6). In the second view, the conserved Arg 286 and Lys 274 , which are in close proximity to the moenomycin phosphonate moiety, directly protonate the donor pyrophosphate leaving group (26). Following glycosyl bond formation, the product must translocate from the acceptor to the donor site.…”

“…5, A and C). The Lys 274 and Arg 286 residues are required for optimal lipid II polymerization activity in the S. aureus monofunctional GTase (26) and by analogy to their interaction with the moenomycin phosphonate are likely important for stabilizing the donor lipid pyrophosphate moiety. Taken together, the EF ring phosphonate portion of moenomycin makes an intricate network of hydrogen bonding interactions with conserved residues in and around the E. coli PBP1b catalytic site that are essential for polymerization of the natural substrate.…”