title: UDP-N-acetylmuramic acid (UDP-MurNAc) is a potent inhibitor of MurA (enolpyruvyl-UDP-GlcNAc synthase).
abstract: Purified recombinant MurA (enolpyruvyl-UDP-GlcNAc synthase) overexpressed in Escherichia coli had significant amounts of UDP-MurNAc (UDP-N-acetylmuramic acid) bound after purification. UDP-MurNAc is the product of MurB, the next enzyme in peptidoglycan biosynthesis. About 25% of MurA was complexed with UDP-MurNAc after five steps during purification that should have removed it. UDP-MurNAc isolated from MurA was identified by mass spectrometry, NMR analysis, and comparison with authentic UDP-MurNAc. Subsequent investigation showed that UDP-MurNAc bound to MurA tightly, with K(d,UDP)(-)(MurNAc) = 0.94 +/- 0.04 microM, as determined by fluorescence titrations using ANS (8-anilino-1-naphthalenesulfonate) as an exogenous fluorophore. UDP-MurNAc binding was competitive with ANS and phosphate, the second product of MurA, and it inhibited MurA. The inhibition patterns were somewhat ambiguous, likely being competitive with the substrate PEP (phosphoenolpyruvate) and either competitive or noncompetitive with respect to the substrate UDP-GlcNAc (UDP-N-acetylglucosamine). These results indicate a possible role for UDP-MurNAc in regulating the biosynthesis of nucleotide precursors of peptidoglycan through feedback inhibition. Previous studies indicated that UDP-MurNAc binding to MurA was not tight enough to be physiologically relevant; however, this was likely an artifact of the assay conditions.
authors.affiliation: Department of Chemistry, Antimicrobial Research Centre, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Shehadeh
authors.initials: S
authors.lastname: Mizyed
authors.forename: Anna
authors.initials: A
authors.lastname: Oddone
authors.forename: Bartosz
authors.initials: B
authors.lastname: Byczynski
authors.forename: Donald W
authors.initials: DW
authors.lastname: Hughes
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Berti
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D010728:Phosphoenolpyruvate / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000817:Anilino Naphthalenesulfonates; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C027132:1-anilino-8-naphthalenesulfonate; D010728:Phosphoenolpyruvate; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase