Identification of a Functionally Unique Family of Penicillin-Binding Proteins

Abstract

Penicillin-binding proteins (PBPs) are enzymes involved in the assembly of the bacterial cell wall, a major target for antibiotics. These proteins are classified by mass into high molecular weight PBPs, which are transpeptidases that form peptidoglycan crosslinks, and low molecular weight PBPs, which are typically hydrolases. We report a functionally unique family of low molecular weight PBPs that act as transpeptidases rather than hydrolases, but they do not crosslink peptidoglycan. We show that these PBPs can exchange D-amino acids bearing chemical tags or affinity handles into peptidoglycan precursors, including Lipid II, enabling biochemical studies of proteins involved in cell wall assembly. We report that, in two organisms, the PBPs incorporate lysine into peptidoglycan and, further, that this linkage can occur via attack of the primary ε-amine side chain, an unprecedented reaction for a PBP.

Promotion of a Reaction by Cooling: Stereoselective 1,2‐cis‐α‐Thioglycoconjugation by Thiol‐Ene Coupling at −80 °C

“…The α‐ O ‐glycosides are abundantly found in nature and many 1,2‐ cis ‐α‐linked sugars including α‐ l ‐fucosides, α‐ d ‐galactosides as well as 2‐deoxy‐2‐aminoglycosides with α‐ d ‐ gluco and α‐ d ‐ galacto configurations play important roles in various biological processes. For example, α‐GlcNAc is a key component of lipid II; a precursor molecule used in the biosynthesis of the peptidoglycan cell wall of bacteria, α‐galactosylceramide is a glycolipid of an important immunostimulant activity, and the α‐ N Ac‐galactosamine linked to serine or threonine represents the conserved region of the mucin O ‐glycoproteins and also known as a tumor‐associated carbohydrate antigen (Tn antigen) . l ‐Fucose (6‐deoxy‐ l ‐galactose), always α‐linked at a terminal position, is a common component of many N ‐ and O ‐glycoproteins and glycolipids produced by mammalian cells and it is involved in a large number of cellular processes .…”

Maturing Mycobacterial Peptidoglycan Requires Non-canonical Crosslinks to Maintain Shape

“…The pH was adjusted to ~4 using with 20% H3PO4 and the resulting product was lyophilized to dryness. The residue was resuspended in 18µL of water and analyzed via LC-MS as previously reported (43). Figure 3C.…”

The cell cycle in Staphylococcus aureus is regulated by an amidase that controls peptidoglycan synthesis

“…4c). To visualize the polymers, we labeled the stem peptides with biotin-D-Lys (BDL) for immunoblotting 19 . No changes in the polymers were observed with LytH treatment alone, but in the presence of the complex, we saw a reduction in the intensity of BDL-labeled glycan strands (Fig.…”

“…Overexpression and purification of PBPX: E. coli BL21(DE3) was transformed with plasmid pMW1010 encoding E. faecalis His 6 - PBPX [T36-P429]. This protein was expressed and purified as reported previously 19 .…”