title: Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase.
abstract: The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after 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 peptidoglycan GTs. In addition, our study indicates that a modification of two residues (L119N 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.
authors.affiliation: Department of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaussstr. 20, 42119 Wuppertal, Germany. Electronic address: bury@ipa-dguv.de.
authors.forename: Daniel
authors.initials: D
authors.lastname: Bury
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Allée de la Chimie, B6a, B-4000, Sart Tilman, Liège, Belgium.
authors.forename: Ismahene
authors.initials: I
authors.lastname: Dahmane
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Allée de la Chimie, B6a, B-4000, Sart Tilman, Liège, Belgium.
authors.forename: Adeline
authors.initials: A
authors.lastname: Derouaux
authors.affiliation: Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
authors.forename: Shrinivas
authors.initials: S
authors.lastname: Dumbre
authors.affiliation: Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
authors.forename: Piet
authors.initials: P
authors.lastname: Herdewijn
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Allée de la Chimie, B6a, B-4000, Sart Tilman, Liège, Belgium.
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.affiliation: Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Eefjan
authors.initials: E
authors.lastname: Breukink
authors.affiliation: Department of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaussstr. 20, 42119 Wuppertal, Germany.
authors.forename: Erika
authors.initials: E
authors.lastname: Mueller-Seitz
authors.affiliation: Department of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaussstr. 20, 42119 Wuppertal, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Petz
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Allée de la Chimie, B6a, B-4000, Sart Tilman, Liège, Belgium. Electronic address: mterrak@ulg.ac.be.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Terrak
mesh_terms: D001455:Bambermycins / Q000378:metabolism; D056265:Microbial Interactions / Q000502:physiology*; D046928:Peptidoglycan Glycosyltransferase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D001455:Bambermycins; C437243:moenomycin A; D046928:Peptidoglycan Glycosyltransferase
keywords: Glycosyltransferase; Lipid II; Moenomycin; Peptidoglycan; SPR