Peptidoglycan glycosyltransferases (GTs) catalyze the polymerization step of cell-wall biosynthesis, are membrane-bound, and are highly conserved across all bacteria. Long considered the "holy grail" of antibiotic research, they represent an essential and easily accessible drug target for antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus. We have determined the 2.8 angstrom structure of a bifunctional cell-wall cross-linking enzyme, including its transpeptidase and GT domains, both unliganded and complexed with the substrate analog moenomycin. The peptidoglycan GTs adopt a fold distinct from those of other GT classes. The structures give insight into critical features of the catalytic mechanism and key interactions required for enzyme inhibition.
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"Though it lost a number of essential genes, it retained those that are supportive in cell wall formation and cell wall processes (Eiglmeier et al. 2001). Glycosyltranfersases play a vital role in cell wall biosynthesis (Lovering et al. 2007). We recognized a number of HPs, such as B8ZTV0, B8ZTW3, B8ZU23, B8ZUC5, B8ZRJ0, showing glycosyltranferase activity. "
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium leprae is an intracellular obligate parasite that causes leprosy in humans, and it leads to the destruction of peripheral nerves and skin deformation. Here, we report an extensive analysis of the hypothetical proteins (HPs) from M. leprae strain Br4923, assigning their functions to better understand the mechanism of pathogenesis and to search for potential therapeutic interventions. The genome of M. leprae encodes 1604 proteins, of which the functions of 632 are not known (HPs). In this paper, we predicted the probable functions of 312 HPs. First, we classified all HPs into families and subfamilies on the basis of sequence similarity, followed by domain assignment, which provides many clues for their possible function. However, the functions of 320 proteins were not predicted because of low sequence similarity with proteins of known function. Annotated HPs were categorized into enzymes, binding proteins, transporters, and proteins involved in cellular processes. We found several novel proteins whose functions were unknown for M. leprae. These proteins have a requisite association with bacterial virulence and pathogenicity. Finally, our sequence-based analysis will be helpful for further validation and the search for potential drug targets while developing effective drugs to cure leprosy.
"Elongation of the growing chain is achieved by the addition of disaccharide unit MurNAc-GlcNAc of lipid II in a processive way. Comparison of the apo and moenomycin A bound structures shows that the head domain remains unchanged after moenomycin A binding whereas a major conformational change occurs in the jaw subdomain with partial restructuration of the mobile region . At the initiation phase of polymerization, the lipid II substrate molecules bind to the donor and acceptor sites which makes binding studies to determine the affinity of the substrate for each site particularly complex. "
[Show abstract][Hide abstract] ABSTRACT: http://www.sciencedirect.com/science/article/pii/S0006295214006601
The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which following cross linking by the transpeptidases form the net-like peptidoglycan macromolecule. The essential function of the GT makes it an attractive antimicrobial target; therefore a better understanding of its function and its mechanism of interaction with substrates could help in the design and the development of new antibiotics.In this work, we have used a surface plasmon resonance Biacore® biosensor, based on an amine derivative of moenomycin A immobilized on a sensor chip surface, to investigate the mechanism of binding of substrate analogous inhibitors to the GT. Addition of increasing concentrations of moenomycin A to the Staphylococcus aureus MtgA led to reduced binding of the protein to the sensor chip as expected. Remarkably, in the presence of low concentrations of the most active disaccharide inhibitors, binding of MtgA to immobilized moenomycin A was found to increase; in contrast competition with moenomycin A occurred only at high concentrations. This finding suggests that at low concentrations, the lipid II analogs bind to the acceptor site and induce a cooperative binding of moenomycin A to the donor site. Our results constitute the first indication of the existence of a positive cooperativity between the acceptor and the donor sites of GTs.In addition, our study indicates that a modification of two residues (L119 N and F120S) within the hydrophobic region of MtgA can yield monodisperse forms of the protein with apparently no change in its secondary structure content, but this is at the expense of the enzyme function.
Full-text · Article · Nov 2014 · Biochemical Pharmacology
"Several analogs have been prepared and characterized, including fluorescently labeled moenomycin, which was used to develop a high-throughput screening assay (Welzel, 2005; Adachi et al., 2006; Cheng et al., 2008). Efforts in protein expression and purification from different bacterial species have allowed the determination of the crystal structures of GTs both in the apo form and in complex with moenomycin or analogs (Lovering et al., 2007; Yuan et al., 2007; Heaslet et al., 2009; Sung et al., 2009). The sequences and structures of the GTs are highly conserved. "
[Show abstract][Hide abstract] ABSTRACT: Peptidoglycan (PG) is an essential net-like macromolecule that surrounds bacteria, gives them their shape, and protects them against their own high osmotic pressure. PG synthesis inhibition leads to bacterial cell lysis, making it an important target for many antibiotics. The final two reactions in PG synthesis are performed by penicillin-binding proteins (PBPs). Their glycosyltransferase (GT) activity uses the lipid II precursor to synthesize glycan chains and their transpeptidase (TP) activity catalyzes the cross-linking of two glycan chains via the peptide side chains. Inhibition of either of these two reactions leads to bacterial cell death. β-lactam antibiotics target the transpeptidation reaction while antibiotic therapy based on inhibition of the GTs remains to be developed. Ongoing research is trying to fill this gap by studying the interactions of GTs with inhibitors and substrate mimics and utilizing the latter as templates for the design of new antibiotics. In this review we present an updated overview on the GTs and describe the structure-activity relationship of recently developed synthetic ligands.
Full-text · Article · Mar 2013 · Frontiers in Immunology