A mechanism-based inhibitor targeting the DD-transpeptidase activity of bacterial penicillin-binding proteins

Penicillin-binding proteins (PBPs) are responsible for the final stages of bacterial cell wall assembly. These enzymes are targets of beta-lactam antibiotics. Two of the PBP activities include dd-transpeptidase and DD-carboxypeptidase activities, which carry out the cross-linking of the cell wall and trimming of the peptidoglycan, the major constituent of the cell wall, by an amino acid, respectively. The activity of the latter enzyme moderates the degree of cross-linking of the cell wall, which is carried out by the former. Both these enzymes go through an acyl-enzyme species in the course of their catalytic events. Compound 6, a cephalosporin derivative incorporated with structural features of the peptidoglycan was conceived as an inhibitor specific for DD-transpeptidases. On acylation of the active sites of dd-transpeptidases, the molecule would organize itself in the two active site subsites such that it mimics the two sequestered strands of the bacterial peptidoglycan en route to their cross-linking. Hence, compound 6 is the first inhibitor conceived and designed specifically for inhibition of DD-transpeptidases. The compound was synthesized in 13 steps and was tested with recombinant PBP1b and PBP5 of Escherichia coli, a dd-transpeptidase and a dd-carboxypeptidase, respectively. Compound 6 was a time-dependent and irreversible inhibitor of PBP1b. On the other hand, compound 6 did not interact with PBP5, neither as an inhibitor (reversible or irreversible) nor as a substrate.     

Transpeptidase-mediated incorporation of D-amino acids into bacterial peptidoglycan

The β-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 cross-linking 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 noncanonical D-amino acids can be introduced into the bacterial cell wall.     

An Activity‐Based Probe for Studying Crosslinking in Live Bacteria

Penicillin‐binding proteins (PBPs) catalyze the crosslinking of peptidoglycan (PG), an essential process for bacterial growth and survival, and a common antibiotic target. Yet, despite its importance, little is known about the spatiotemporal aspects of crosslinking—largely because of a lack of experimental tools for studying the reaction in live bacteria. Here we introduce such a tool: an activity‐based probe that enables visualization and relative quantitation of crosslinking in vivo. In Staphylococcus aureus, we show that fluorescent mimics of the natural substrate of PBPs (PG stem peptide) are covalently incorporated into the cell wall, installing fluorophores in place of natural crosslinks. These fluorescent stem peptide mimics (FSPMs) are selectively recognized by a single PBP in S. aureus: PBP4. Thus, we were able to use FSPM pulse‐labeling to localize PBP4 activity in live cells, showing that it is recruited to the septum in a manner dependent on wall teichoic acid.     

Lytic transglycosylase MltB of Escherichia coli and its role in recycling of peptidoglycan strands of bacterial cell wall

The cell wall is an indispensable structure for the survival of bacteria and a target for antibiotics. Peptidoglycan is the major constituent of the cell wall, which is comprised of backbone repeats of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). A peptide stem is appended to the NAM unit, which in turn experiences cross-linking with a peptide from another peptidoglycan in the final steps of cell wall assembly. In the normal course of bacterial growth, as much as 60% of the parental cell wall is recycled, a process that is not fully understood. A polymeric cell wall is fragmented by the family of lytic transglycosylases, and certain key fragments are transported to the cytoplasm for recycling. The genes for the six known lytic transglycosylases of Escherichia coli were cloned, and the enzymes were purified in this study. It is shown that MltB is the only lytic transglycosylase to turn over a synthetic peptidoglycan fragment of two NAG-NAM repeats; hence this enzyme is likely to be the lytic transglycosylase responsible for processing of shorter peptidoglycan strands. Lytic transglycosylases have been proposed to go through an oxocarbenium species that would trap the 6-hydroxyl moiety of the glucosamine residue of muramic acid to generate the so-called 1,6-anhydromuramyl moiety. It is documented herein by characterization of the products of turnover that this process takes place to the total exclusion of the entrapment of a water molecule by the reactive intermediary oxocarbenium species. Furthermore, turnover of the E. coli sacculus (whole cell wall) by MltB was characterized. It is documented that each MltB molecule is able to process the cell wall 14000 times in the course of a single doubling time for E. coli.     

In Vivo Probe of Lipid II‐Interacting Proteins

β‐Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin‐binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP–Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d ‐amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.     

Co-opting the cell wall in fighting methicillin-resistant Staphylococcus aureus: potent inhibition of PBP 2a by two anti-MRSA beta-lactam antibiotics

Methicillin-resistant Staphylococcus aureus (MRSA) is a global bacterial scourge that has become resistant to many classes of antibiotics, and treatment options for MRSA infections are limited. The cause of MRSA resistance to all commercially available beta-lactam antibiotics is the acquisition of the gene mecA, which encodes penicillin-binding protein 2a (PBP 2a). PBP 2a is a transpeptidase, which in contrast to the other transpeptidases of S. aureus does not experience inhibition by beta-lactam antibiotics. The lack of inhibition is due to a closed conformation for the active site for PBP 2a, which opens up only in the course of the catalytic function of the protein. Here we show that two new anti-MRSA antibiotics now undergoing clinical trials, ceftaroline and ME1036, are able to inhibit PBP 2a effectively, a process that is enhanced in the presence of a cell wall structural surrogate. It is likely that in the course of bacterial growth the occupancy of the allosteric site for the cell wall is co-opted by these antibiotics, and under these conditions the second-order rate constant for the encounter of the antibiotic and PBP 2a approaches the clinically useful value of 10(4)-10(5) M-1 s-1. These compounds are potent inhibitors of PBP 2a as well as PBPs from other species, and have potential as therapeutic agents for treatment of serious infections by MRSA and other resistant bacterial pathogens.     

Structural aspects for evolution of beta-lactamases from penicillin-binding proteins

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and beta-lactamases, resistance enzymes to beta-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for beta-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7beta-[N-Acetyl-L-alanyl-gamma-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC beta-lactamase from Escherichia coli was solved at 1.71 A resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the -lactamase active site. Furthermore, insertion of a peptide in the beta-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the beta-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.     

Differential inhibition of Staphylococcus aureus PBP2 by glycopeptide antibiotics

The glycopeptide antibiotics prevent maturation of the bacterial cell wall by binding to the terminal d-alanyl-d-alanine moiety of peptidoglycan precursors, thereby inhibiting the enzymes involved in the final stages of peptidoglycan synthesis. However, there are significant differences in the biological activity of particular glycopeptide derivatives that are not related to their affinity for d-Ala-d-Ala. We compare the ability of vancomycin and a set of clinically relevant glycopeptides to inhibit Staphylococcus aureus PBP2 (penicillin binding protein), the major transglycosylase in a clinically relevant pathogen, S. aureus. We report experiments suggesting that activity differences between glycopeptides against this organism reflect a combination of substrate binding and secondary interactions with key enzymes involved in peptidoglycan synthesis.     

Mixed quantum mechanical/molecular mechanical (QM/MM) study of the deacylation reaction in a penicillin binding protein (PBP) versus in a class C beta-lactamase

The origin of the substantial difference in deacylation rates for acyl-enzyme intermediates in penicillin-binding proteins (PBPs) and beta-lactamases has remained an unsolved puzzle whose solution is of great importance to understanding bacterial antibiotic resistance. In this work, accurate, large-scale mixed ab initio quantum mechanical/molecular mechanical (QM/MM) calculations have been used to study the hydrolysis of acyl-enzyme intermediates formed between cephalothin and the dd-peptidase of Streptomyces sp. R61, a PBP, and the Enterobacter cloacae P99 cephalosporinase, a class C beta-lactamase. Qualitative and, in the case of P99, quantitative agreement was achieved with experimental kinetics. The faster rate of deacylation in the beta-lactamase is attributed to a more favorable electrostatic environment around Tyr150 in P99 (as compared to that for Tyr159 in R61) which facilitates this residue's function as the general base. This is found to be in large part accomplished by the ability of P99 to covalently bind the ligand without concurrent elimination of hydrogen bonds to Tyr150, which proves not to be the case with Tyr159 in R61. This work provides an essential foundation for further work in this area, such as selecting mutations capable of converting the PBP into a beta-lactamase.     

Activation for catalysis of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by bacterial cell wall

Methicillin-resistant Staphylococcus aureus (MRSA) has acquired a unique penicillin-binding protein (PBP), PBP 2a, which has rendered the organism resistant to the action of all available beta-lactam antibiotics. The X-ray structure of PBP 2a shows the active site in a closed conformation, consistent with resistance to inhibition by beta-lactam antibiotics. However, it is known that PBP 2a avidly cross-links the S. aureus cell wall, which is its physiological function. It is shown herein that synthetic fragments of the bacterial cell wall bind in a saturable manner to PBP 2a and cause a conformational change in the protein that makes the active site more accessible to binding to a beta-lactam antibiotic. These observations and measurements point to a novel strategy by nature to keep the active site of PBP 2a sheltered from the inhibitory activity of the antibiotics, yet it becomes available to the polymeric cell wall by a requisite conformational change for the critical cell wall cross-linking reaction.     

Synthesis of modified peptidoglycan precursor analogues for the inhibition of glycosyltransferase

The peptidoglycan glycosyltransferases (GTs) are essential enzymes that catalyze the polymerization of glycan chains of the bacterial cell wall from lipid II and thus constitute a validated antibacterial target. Their enzymatic cavity is composed of a donor site for the growing glycan chain (where the inhibitor moenomycin binds) and an acceptor site for lipid II substrate. In order to find lead inhibitors able to fill this large active site, we have synthesized a series of substrate analogues of lipid I and lipid II with variations in the lipid, the pyrophosphate, and the peptide moieties and evaluated their biological effect on the GT activity of E. coli PBP1b and their antibacterial potential. We found several compounds able to inhibit the GT activity in vitro and cause growth defect in Bacillus subtilis . The more active was C16-phosphoglycerate-MurNAc-(L-Ala-D-Glu)-GlcNAc, which also showed antibacterial activity. These molecules are promising leads for the design of new antibacterial GT inhibitors.     

Lipid II: total synthesis of the bacterial cell wall precursor and utilization as a substrate for glycosyltransfer and transpeptidation by penicillin binding protein (PBP) 1b of Escherichia coli.

An essential feature in the life cycle of both gram positive and gram negative bacteria is the production of new cell wall. Also known as murein, the cell wall is a two-dimensional polymer, consisting of a linear, repeating N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) motif, cross-linked via peptides appended to MurNAc. The final steps in the maturation of murein are catalyzed by a single, bifunctional enzyme, known as a high MW, class A penicillin binding protein (PBP). PBPs catalyze polymerization of the sugar units (glycosyltransfer), as well as peptide cross-linking (transpeptidation) utilizing Lipid II as substrate. Detailed enzymology on this enzyme has been limited, due to difficulties in obtaining sufficient amounts of Lipid II, as well as the availability of a convenient and informative assay. We report the total chemical synthesis of Lipid II, as well as the development of an appropriate assay system and the observation of both catalytic transformations.     

Staphylococcus aureus Penicillin‐Binding Protein 2 Can Use Depsi‐Lipid II Derived from Vancomycin‐Resistant Strains for Cell Wall Synthesis

Vancomycin‐resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide‐containing modified cell‐wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin‐binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild‐type S. aureus. However, it is unclear whether VRSA processes the depsipeptide‐containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi‐lipid I, a cell‐wall precursor of VRSA. By using this chemistry, we prepared a depsi‐lipid II analogue as substrate for a cell‐free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi‐lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.     

A computational evaluation of the mechanism of penicillin-binding protein-catalyzed cross-linking of the bacterial cell wall

Penicillin-binding protein 1b (PBP 1b) of the gram-positive bacterium Streptococcus pneumoniae catalyzes the cross-linking of adjacent peptidoglycan strands, as a critical event in the biosynthesis of its cell wall. This enzyme is representative of the biosynthetic PBP structures of the β-lactam-recognizing enzyme superfamily and is the target of the β-lactam antibiotics. In the cross-linking reaction, the amide between the -D-Ala-D-Ala dipeptide at the terminus of a peptide stem acts as an acyl donor toward the ε-amino group of a lysine found on an adjacent stem. The mechanism of this transpeptidation was evaluated using explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics calculations. Sequential acyl transfer occurs to, and then from, the active site serine. The resulting cross-link is predicted to have a cis-amide configuration. The ensuing and energetically favorable cis- to trans-amide isomerization, within the active site, may represent the key event driving product release to complete enzymatic turnover.     

Thermodynamics of interactions of vancomycin and synthetic surrogates of bacterial cell wall

Glycopeptide antibiotics, including vancomycin, form complexes via a set of five hydrogen bonds with the acyl-l-Lys-d-Ala-d-Ala portion of the peptidyl stems of the bacterial cell wall peptidoglycan. This complexation deprives the organism from the ability to cross-link peptidyl stems of the peptidoglycan, leading to bacterial cell death. Four synthetic fragments as surrogates of the components of the bacterial cell wall have been prepared in our lab in multistep syntheses. These synthetic samples were used in investigations of the thermodynamics properties (DeltaG degrees , DeltaH degrees , and TDeltaS degrees ) for the complexation with vancomycin by isothermal titration calorimetry (ITC). Complexation with the glycopeptide analogues is largely enthalpy-driven (formation of five hydrogen bonds), and in the analogues with a single peptidyl stem, the complexation is 1:1. The complexation is more complicated with an approximately 2 kDa cell wall surrogate (compound 4), which possesses two peptidyl stems. The data were suggestive of interactions between the two vancomycin molecules, with an entropic penalty attributable to restriction of molecular movements within the complex due to restriction of motion of the highly mobile acyl-d-Ala-d-Ala moiety of the peptidyl stems. These data were reconciled with the recently determined NMR solution structure for the peptidoglycan fragment 4 and its implications for the larger cell wall.     

Acylation and deacylation mechanism and kinetics of penicillin G reaction with Streptomyces R61 DD-peptidase

Two quantum mechanical (QM)-cluster models are built for studying the acylation and deacylation mechanism and kinetics of Streptomyces R61 DD-peptidase with the penicillin G at atomic level detail. DD-peptidases are bacterial enzymes involved in the cross-linking of peptidoglycan to form the cell wall, necessary for bacterial survival. The cross-linking can be inhibited by antibiotic beta-lactam derivatives through acylation, preventing the acyl-enzyme complex from undergoing further deacylation. The deacylation step was predicted to be rate-limiting. Transition state and intermediate structures are found using density functional theory in this study, and thermodynamic and kinetic properties of the proposed mechanism are evaluated. The acyl-enzyme complex is found lying in a deep thermodynamic sink, and deacylation is indeed the severely rate-limiting step, leading to suicide inhibition of the peptidoglycan cross-linking. The usage of QM-cluster models is a promising technique to understand, improve, and design antibiotics to disrupt function of the Streptomyces R61 DD-peptidase.     

The first total synthesis of lipid II: the final monomeric intermediate in bacterial cell wall biosynthesis

Bacterial peptidoglycan is composed of a network of beta-[1,4]-linked glyan strands that are cross-linked through pendant peptide chains. The final product, the murein sacculus, is a single, covalently closed macromolecule that precisely defines the size and shape of the bacterial cell. The recent increase in bacterial resistance to cell wall active agents has led to a resurgence of activity directed toward improving our understanding of the resistance mechanisms at the molecular level. The biosynthetic enzymes and their natural substrates can be invaluable tools in this endeavor. While modern experimental techniques have led to isolation and purification of the biosynthetic enzymes utilized in peptidoglycan biosynthesis, securing useful quantities of their requisite substrates from natural substrates has remained problematic. In an effort to address this issue, we report the first total synthesis of lipid II (4), the final monomeric intermediate utilized by Gram positive bacteria for peptidoglycan biosynthesis.     

Peptidoglycan as Nod1 ligand; fragment structures in the environment, chemical synthesis, and their innate immunostimulation

Covering: up to 2011. This review focuses on the recent revealing of the immunostimulatory bacterial cell wall peptidoglycan (PGN) fragments as Nod1 ligands, especially a newly developed chemical synthesis of the partial structures, fragment structures in the environment and bacterial supernatant, and the immunostimulatory activities of the Nod1 ligands.     

Isolated peptidoglycan glycosyltransferases from different organisms produce different glycan chain lengths

Peptidoglycan is an essential component of bacterial cell wall. The glycan strands of peptidoglycan are synthesized by enzymes called peptidoglycan glycosyltransferases (PGTs). Using a high-resolution 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.     

Potentiometric phosphate-sensing system utilizing phosphate-binding protein

A phosphate-detection system has been developed which uses phosphate-binding protein (PBP) from Escherichia coli. PBP was immobilized on a sheet of nitrocellulose membrane by cross-linking and the membrane potential of the immobilized PBP was measured. The response time of the system to phosphate was 5 min. The response was selective to phosphate among other anions. Under optimum conditions 0.1-1.5 mmol L(-1) phosphate can be determined with this system.