title: Forming cross-linked peptidoglycan from synthetic gram-negative Lipid II.
abstract: The bacterial cell wall precursor, Lipid II, has a highly conserved structure among different organisms except for differences in the amino acid sequence of the peptide side chain. Here, we report an efficient and flexible synthesis of the canonical Lipid II precursor required for the assembly of Gram-negative peptidoglycan (PG). We use a rapid LC/MS assay to analyze PG glycosyltransfer (PGT) and transpeptidase (TP) activities of Escherichia coli penicillin binding proteins PBP1A and PBP1B and show that the native m-DAP residue in the peptide side chain of Lipid II is required in order for TP-catalyzed peptide cross-linking to occur in vitro. Comparison of PG produced from synthetic canonical E. coli Lipid II with PG isolated from E. coli cells demonstrates that we can produce PG in vitro that resembles native structure. This work provides the tools necessary for reconstituting cell wall synthesis, an essential cellular process and major antibiotic target, in a purified system.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Lebar
authors.forename: Tania J
authors.initials: TJ
authors.lastname: Lupoli
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsukamoto
authors.forename: Janine M
authors.initials: JM
authors.lastname: May
authors.forename: Suzanne
authors.initials: S
authors.lastname: Walker
authors.forename: Daniel
authors.initials: D
authors.lastname: Kahne
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000378:metabolism*; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D046928:Peptidoglycan Glycosyltransferase / Q000378:metabolism*; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000378:metabolism*; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; D046928:Peptidoglycan Glycosyltransferase; C575488:penicillin-binding protein 1A, E coli; C478681:penicillin-binding protein 1B, E coli; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase