Continuous Fluorescence Assay for Peptidoglycan Glycosyltransferases.
Bacterial cell wall peptidoglycan is synthesized from its precursor lipid II by two enzymatic reactions. First, glycosyltransferases polymerize the glycan strands and second, DD-transpeptidases form cross-links between peptides of neighboring strands. Most bacteria possess bifunctional peptidoglycan synthesis enzymes capable of catalyzing both reactions. Here, we describe a continuous fluorescence glycosyltransferase assay using Dansyl-labeled lipid II as substrate. Progression of the reaction is monitored by the reduction in fluorescence over time. The assay is suitable to investigate the effect of protein interaction partners on the glycan strand synthesis activity of peptidoglycan polymerases.
Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK. alexander.egan@ncl.ac.uk.
Alexander J F
AJ
Egan
Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK. w.vollmer@ncl.ac.uk.
Waldemar
W
Vollmer
D002473:Cell Wall / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry; D010457:Peptidoglycan / Q000096:biosynthesis; D046928:Peptidoglycan Glycosyltransferase / Q000737:chemistry / Q000302:isolation & purification*
D005456:Fluorescent Dyes; D010457:Peptidoglycan; D046928:Peptidoglycan Glycosyltransferase
Continuous fluorescence assay; Glycosyltransferase; Peptidoglycan; Peptidoglycan synthesis; Synthase