The beta-lactams are the most important class of antibiotics in clinical use. Their lethal targets are the transpeptidase domains of penicillin binding proteins (PBPs), which catalyze the crosslinking of bacterial peptidoglycan (PG) during cell wall synthesis. The transpeptidation reaction occurs in two steps, the first being formation of a covalent enzyme intermediate and the second involving attack of an amine on this intermediate. Here we use defined PG substrates to dissect the individual steps catalyzed by a purified E. coli transpeptidase. We demonstrate that this transpeptidase accepts a set of structurally diverse D-amino acid substrates and incorporates them into PG fragments. These results provide new information on donor and acceptor requirements as well as a mechanistic basis for previous observations that non-canonical D-amino acids can be introduced into the bacterial cell wall.

Bacterial cells are surrounded by a cross-linked polymer called peptidoglycan, the integrity of which is necessary for cell survival. The carbohydrate chains that form the backbone of peptidoglycan are made by peptidoglycan glycosyltransferases (PGTs), highly conserved membrane-bound enzymes that are thought to be excellent targets for the development of new antibacterials. Although structural information on these enzymes recently became available, their mechanism is not well understood because of a dearth of methods to monitor PGT activity. Here we describe a direct, sensitive, and quantitative SDS-PAGE method to analyze PGT reactions. We apply this method to characterize the substrate specificity and product length profile for two different PGT domains, PBP1A from Aquifex aeolicus and PBP1A from Escherichia coli. We show that both disaccharide and tetrasaccharide diphospholipids (Lipid II and Lipid IV) serve as substrates for these PGTs, but the product distributions differ significantly depending on which substrate is used as the starting material. Reactions using the disaccharide substrate are more processive and yield much longer glycan products than reactions using the tetrasaccharide substrate. We also show that the SDS-PAGE method can be applied to provide information on the roles of invariant residues in catalysis. A comprehensive mutational analysis shows that the biggest contributor to turnover of 14 mutated residues is an invariant glutamate located in the center of the active site cleft. The assay and results described provide new information about the process by which PGTs assemble bacterial cell walls.

Bacteria are surrounded by a cell wall containing layers of peptidoglycan, the integrity of which is essential for bacterial survival. In the final stage of peptidoglycan biosynthesis, peptidoglycan glycosyltransferases (PGTs; also known as transglycosylases) catalyze the polymerization of Lipid II to form linear glycan chains. PGTs have tremendous potential as antibiotic targets, but the potential has not yet been realized. Mechanistic studies have been hampered by a lack of substrates to monitor enzymatic activity. We report here the total synthesis of heptaprenyl-Lipid IV and its use to study two different PGTs from E. coli. We show that one PGT can couple Lipid IV to itself whereas the other can only couple Lipid IV to Lipid II. These in vitro differences in enzymatic activity may reflect differences in the biological functions of the two major glycosyltransferases in E coli.

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.Peptidoglycan glycosyltransferases (PGTs) are highly conserved bacterial enzymes that catalyze the polymerization of a disaccharide called Lipid II (Figure 1) to form the glycan strands of peptidoglycan. PGTs are regarded as desirable antibiotic targets because they are extracellular, they do not have eukaryotic counterparts, and they play an essential role in a validated therapeutic pathway. 1 Although their importance has been appreciated for decades, the mechanism of glycan chain polymerization is not yet understood. One aspect of the PGT reaction that is central to defining the mechanism is the direction of glycan chain elongation ( Figure 2A). 2 Here, we describe an approach to test the direction of glycan chain growth. We have applied this strategy to four different PGTs from both Gram-negative and Grampositive organisms, including two PGTs for which crystal structures were recently reported. 3 The results show that all these PGTs polymerize Lipid II by the addition of new disaccharide units to the anomeric (diphospholipid) end of the elongating polymer (called the "reducing end" by convention, although not a lactol).Our strategy to determine the direction of glycan polymerization, illustrated in Figure 2B, relies on the synthesis of nascent peptidoglycan chains that are blocked at their non-reducing ends ( Figure 2B). If elongation occurs by reaction at the reducing end of the growing polymer, PGTs should elongate these substrates in the presence of Lipid II ( Figure 2B NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript right). If elongation occurs in the other direction, then these blocked oligomers will not be substrates for PGTs ( Figure 2B, left).To enable our strategy for probing the direction of chain elongation, we required a facile method to selectively modify the non-reducing ends of the nascent glycan chains. We chose to utilize β-1,4-galactosyltransferase (GalT), an enzyme that transfers galactose (Gal) from UDP-Gal ...

Peptidoglycan is an essential component of bacterial cell wall. The glycan strands of peptidoglycan are synthesized by enzymes called peptidoglycan glycosyltransferases (PGTs). Using a highresolution SDS-PAGE assay, we compared the glycan strand lengths of four different PGTs from three different organisms (Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus). We report that each enzyme makes a polymer having an intrinsic characteristic length that is independent of the enzyme:substrate ratio. The glycan strand lengths vary considerably depending on the enzyme. These results indicate that each enzyme must have some mechanism, as yet unknown, for controlling product length. The observation that different PGTs produce different length glycan chains may have implications for their cellular roles and for the three dimensional structure of bacterial peptidoglycan.Bacterial cells are surrounded by a polymer matrix comprising crosslinked strands of peptidoglycan (PG). This matrix, called the sacculus, functions as an exoskeleton, maintaining cell shape and enabling the plasma membrane to withstand high internal osmotic pressures. 1 The three dimensional architecture of PG is not yet clear, but is presumed to depend, among other things, on the lengths of the glycan strands, which are synthesized by processive enzymes called peptidoglycan glycosyltransferases (PGTs). 2 Numerous studies have evaluated lengths of glycan strands from digested bacterial sacculi, and a range of values have been reported even for digests from the same organism. 3 Because substrates and appropriate analytical methods were not available until recently, there have been no systematic studies comparing the lengths of glycan strands produced by different PGTs in vitro. Using a high-resolution gel electrophoresis assay recently developed in our laboratory, 2b we report here a comparative study of the glycan strand length distributions produced by four different PGTs, Escherichia coli PBP1A (E. coli PBP1A), Escherichia coli PBP1B (E. coli PBP1B), Enterococcus faecalis PBP2A (E. faecalis PBP2A), and Staphylococcus aureus PBP2 (S. aureus PBP2) (Fig 1). We show that different PGTs produce glycan chains having a characteristic intrinsic length distribution. The intrinsic lengths are a function of the particular PGT but are independent of enzyme:substrate ratios. There is a correlation between the intrinsic in vitro product lengths kahne@chemistry.harvard.edu; suzanne_walker@hms.harvard.edu. and the longest strands isolated from sacculi. The implications of these observations for the architecture of the bacterial cell wall are discussed. NIH Public AccessThe four PGTs we studied were overexpressed, purified and subjected under similar conditions to reaction with heptaprenyl-[ 14 C]-Lipid II (1) (Fig 1a). 2,4,5,6 Unexpectedly, we found that the four PGTs produced glycan chains of different limiting lengths (i.e., the size beyond which the length does not increase even if reaction times are extended and additional substrate is added) ...