title: A microplate assay for the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins; a vancomycin-neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis.
abstract: A microplate, scintillation proximity assay to measure the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[(3)H]N-acetylglucosamine in the presence of 40 μM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-D-ala-D-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a 'neutral' combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.
authors.affiliation: Dept. of Pharmacology, Yale University, CT, USA. Electronic address: vidya.kumar@yale.edu.
authors.forename: Vidya P
authors.initials: VP
authors.lastname: Kumar
authors.affiliation: Duke-NUS Gradate Medical School, Singapor. Electronic address: chandrakala@duke-nus.edu.sg.
authors.forename: Chandrakala
authors.initials: C
authors.lastname: Basavannacharya
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India. Electronic address: sunitadesousa@gmail.com.
authors.forename: Sunita M
authors.initials: SM
authors.lastname: de Sousa
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation; D004347:Drug Interactions; D004789:Enzyme Activation; D004867:Equipment Design; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D008904:Miniaturization; D046915:Penicillin-Binding Proteins / Q000378:metabolism*; D010406:Penicillins / Q000008:administration & dosage*; D010455:Peptides / Q000008:administration & dosage; D010457:Peptidoglycan / Q000096:biosynthesis*; D046928:Peptidoglycan Glycosyltransferase / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000295:instrumentation; D014640:Vancomycin / Q000008:administration & dosage*
chemical_list: D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010455:Peptides; D010457:Peptidoglycan; D014640:Vancomycin; D010458:Peptidyl Transferases; D046928:Peptidoglycan Glycosyltransferase
keywords: PBP1b; Penicillin; Peptidoglycan synthesis; Transglycosylase; Transpeptidase; Vancomycin