title: Observation of the time-course for peptidoglycan lipid intermediate II polymerization by Staphylococcus aureus monofunctional transglycosylase.
abstract: The polymerization of lipid intermediate II by the transglycosylase activity of penicillin-binding proteins (PBPs) represents an important target for antibacterial action, but limited methods are available for quantitative assay of this reaction, or screening potential inhibitors. A new labelling method for lipid II polymerization products using Sanger's reagent (fluoro-2,4-dinitrobenzene), followed by gel permeation HPLC analysis, has permitted the observation of intermediate polymerization products for Staphylococcus aureus monofunctional transglycosylase MGT. Peak formation is inhibited by 6 µM ramoplanin or enduracidin. Characterization by mass spectrometry indicates the formation of tetrasaccharide and octasaccharide intermediates, but not a hexasaccharide intermediate, suggesting a dimerization of a lipid-linked tetrasaccharide. Numerical modelling of the time-course data supports a kinetic model involving addition to lipid-linked tetrasaccharide of either lipid II or lipid-linked tetrasaccharide. Observation of free octasaccharide suggests that hydrolysis of the undecaprenyl diphosphate lipid carrier occurs at this stage in peptidoglycan transglycosylation.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Darren
authors.initials: D
authors.lastname: Braddick
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Sandhu
authors.affiliation: School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: David I
authors.initials: DI
authors.lastname: Roper
authors.affiliation: School of Engineering, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Chappell
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Timothy D H
authors.initials: TDH
authors.lastname: Bugg
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010457:Peptidoglycan / Q000737:chemistry* / Q000378:metabolism; D058105:Polymerization; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology*; D013997:Time Factors; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; D016695:Glycosyltransferases