title: Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5.
abstract: UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
authors.forename: James A
authors.initials: JA
authors.lastname: Schouten
authors.forename: Sangeev
authors.initials: S
authors.lastname: Bagga
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Lloyd
authors.forename: Gianfranco
authors.initials: G
authors.lastname: de Pascale
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Dowson
authors.forename: David I
authors.initials: DI
authors.lastname: Roper
authors.forename: Timothy D H
authors.initials: TD
authors.lastname: Bugg
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D007202:Indicators and Reagents / Q000032:analysis*; D008025:Ligases / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D015394:Molecular Structure; D010453:Peptide Synthases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010457:Peptidoglycan / Q000096:biosynthesis*; D014166:Transferases / Q000737:chemistry; D017855:Transferases (Other Substituted Phosphate Groups); D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D008055:Lipids; D010455:Peptides; D010457:Peptidoglycan; D014530:Uridine Diphosphate; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria; D008025:Ligases; D010453:Peptide Synthases; C122181:VanD protein, Enterococcus faecium; D003545:Cysteine