Drug-resistant bacteria have caused serious medical problems in recent years, and the need for new antibacterial agents is undisputed. Transglycosylase, a multidomain membrane protein essential for cell wall synthesis, is an excellent target for the development of new antibiotics. Here, we determined the X-ray crystal structure of the bifunctional transglycosylase penicillin-binding protein 1b (PBP1b) from Escherichia coli in complex with its inhibitor moenomycin to 2.16-Å resolution. In addition to the transglycosylase and transpeptidase domains, our structure provides a complete visualization of this important antibacterial target, and reveals a domain for protein-protein interaction and a transmembrane helix domain essential for substrate binding, enzymatic activity, and membrane orientation.antibacterial development ͉ antibiotic resistance ͉ membrane protein structure ͉ peptidoglycan synthesis ͉ protein-protein interaction I n the last decade, the prevalence and occasional outbreaks of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), have posed appalling hurdles in the treatment of bacterial infections (1, 2). New antibacterial agents are, as a result, in desperate demand to combat these pernicious antibiotic-resistant problems that can otherwise cause life-or-death struggles.Bacteria cell wall is a mesh-like structure of cross-linked peptidoglycan, which is essential to scaffold the cytoplasmic membrane and to maintain structural integrity of the cell (3). Cell wall synthesis at the membrane surface is mainly carried out by the membrane-bound enzymes, transpeptidases and transglycosylases, and inhibitors of the transpeptidase are among the most popular antibiotics in clinical use today (3).Escherichia coli PBP1b is a bifunctional transglycosylase, also known as peptidoglycan glycosyltransferase or murein synthase. It contains a transmembrane (TM) helix, 2 enzymatic domains [transglycosylase (TG) and transpeptidase (TP)] (4), and a domain composed of Ϸ100 aa residues between TM and TG with unknown structure and functionality (Fig. 1B). For Ͼ50 years, TP has been the main target for 2 most important classes of antibiotics: ␤-lactams (e.g., penicillin and methicillin) and glycopeptides (e.g., vancomycin). Not too long after they were introduced, resistant bacteria had emerged rapidly and caused serious medical problems. In contrast, resistant strains against moenomycin, the only natural inhibitor to TG from Streptomyces, have rarely been found. The development of new antibiotics against TG has been highly anticipated (5), and not until recently have the molecular structures of TG been available, even with the TM structure undefined.Two crystal structures of transglycosylase, a bifunctional transglycosylase from S. aureus (referred to as SaPBP2) and a transglycosylase domain from Aquifex aeolicus (referred to as AaPGT), have been determined recently with their TM domain or TM and TP domains removed, respectively (6-8). These structur...

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...

No abstract