Domain requirement of moenomycin binding to bifunctional transglycosylases and development of high-throughput discovery of antibiotics

Abstract

Moenomycin inhibits bacterial growth by blocking the transglycosylase activity of class A penicillin-binding proteins (PBPs), which are key enzymes in bacterial cell wall synthesis. We compared the binding affinities of moenomycin A with various truncated PBPs by using surface plasmon resonance analysis and found that the transmembrane domain is important for moenomycin binding. Full-length class A PBPs from 16 bacterial species were produced, and their binding activities showed a correlation with the antimicrobial activity of moenomycin against Enterococcus faecalis and Staphylococcus aureus. On the basis of these findings, a fluorescence anisotropy-based high-throughput assay was developed and used successfully for identification of transglycosylase inhibitors.penicillin-binding proteins ͉ transglycosylase inhibitors ͉ fluorescence anisotropy M any common bacterial pathogens, such as Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, have become multidrug-resistant and have emerged as a public health concern, creating an urgent need for new antibiotics.The bacterial cell wall, or its peptidoglycan synthesis pathway, has been targeted for the development of antibacterial agents (1). The synthesis of peptidoglycan consists of several steps, including the formation of lipid I and lipid II, followed by the final transglycosylation and transpeptidation of lipid II to form peptidoglycan (1, 2). Many current antibiotics are ␤-lactam derivatives that target transpeptidation. To our knowledge, no medicines have yet been developed to inhibit the transglycosylation process. The only known potent inhibitors for transglycosylase (TG) are moenomycin complexes (flavomycin), including moenomycin A (Moe A) (Fig. 1A, compound 1), A12, C1, C3, and C4 (3, 4). Among these, Moe A is the most abundant agent in its family (3, 4). The unique antibacterial properties of Moe A have prompted chemists to synthesize moenomycin fragments and derivatives (5-7) in an attempt to develop new antibiotics. Recently, the total synthesis of Moe A (8), and of its biosynthesis pathway (9), has been reported. However, due to poor bioavailability, flavomycin is currently used only as a growth promoter in animal feeds (10).The characterization of class A penicillin-binding proteins (PBPs) and the identification of TG inhibitors require functional PBP and lipid II as the substrate for the enzyme. However, the limited availability of lipid II has hampered the development of effective enzymatic assays for identification of inhibitors. As a result, the majority of the screening methods that are used to search for TG inhibitors, including the low-throughput methods that use surface plasmon resonance (SPR) (12) or radioactive assays (13-16), rely mainly on moenomycin (11). Development of a TG activity assay that is amenable to high-throughput screening (HTS) is thus desirable for inhibitor identification.In this study, we compared the binding of moenomycin to various truncated PBPs and concluded that the transmembrane (TM) domain is c...

Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli

“…We have previously observed that the binding affinity of moenomycin to E. coli PBP1b is TM domain dependent (9). However, no direct interaction between moenomycin and the TM helix was observed in our crystal structure.…”

“…2 B and C). The resemblance between our structure and SaPBP2 may explain the observation that transglycosylases from E. coli and S. aureus share comparable binding affinity to moenomycin (9). In addition, the interacting residues of the TG domain around the E ring, the F ring, the phosphate group, and the carboxylate group of moenomycin are more conserved than the interacting residues with the remaining parts ( Fig.…”

“…These structures revealed critical interactions between protein and moenomycin and served as good platforms for antibiotic development. We have previously demonstrated that the TM helix domain is important for the binding between E. coli PBP1b and moenomycin (9). In addition, we found that the full-length PBP1b also showed a substantially higher TG enzymatic activity than a TM truncated counterpart [supporting information (SI) Fig.…”

“…Several compounds with molecular weights smaller than moenomycin have been reported to compete with the moenomycin and bind directly to the transglycosylase domain (9,13). Although the inhibition efficiencies of these compounds to bacteria are lower than that of moenomycin, the structural information between these compounds and transglycosylase can be studied via molecular modeling or by X-ray crystallography using current E. coli PBP1b structure as a template for structurebased drug design.…”

Bioactive oligosaccharide natural products

“…78 This theory is supported by the fact that the transmembrane portion of PBPs is necessary for moenomycin binding. 80 …”