title: Development of a novel fluorescent substrate for Autolysin E, a bacterial type II amidase.
abstract: The bifunctional Autolysin E from Staphylococcus epidermidis, contains a Zn(2+)-dependent N-acetylmuramoyl-L-alanine amidase AmiE (EC 3.5.1.28). This enzyme hydrolyzes the amide bond between the carbohydrate chain and the peptide stem of bacterial peptidoglycan. Since peptidoglycan is the mayor component of bacterial cell walls, type II amidases like Autolysin E play an essential role in the bacterial life cycle. Therefore bacterial amidases are appropriate drug targets in the development of antibiotics. The drug discovery process relies on sensitive enzyme assay systems to test possible lead candidates for enzyme inhibition. However, specific determination of bacterial amidase activity is complicated because a simple and accurate enzyme assay is currently unavailable. In this study we developed a sensitive fluorescent substrate for the type II amidase Autolysin E from S. epidermidis, which is suitable for quantifying amidase activity in a high throughput compatible fashion. Using derivatives of the substrate Mca-Ala-D-isoGln-Lys(Dnp)-D-Ala-Arg-OH, we were further able to characterize the amidase substrate specificity of Autolysin E.
authors.affiliation: Interfaculty Institute for Biochemistry, University of Tübingen, Ob dem Himmelreich 7, 72074 Tübingen, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lützner
authors.forename: Bernhard
authors.initials: B
authors.lastname: Pätzold
authors.forename: Sebastian
authors.initials: S
authors.lastname: Zoll
authors.forename: Thilo
authors.initials: T
authors.lastname: Stehle
authors.forename: Hubert
authors.initials: H
authors.lastname: Kalbacher
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000032:analysis* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry*; D013212:Staphylococcus epidermidis / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: C539914:7-methoxycoumarin-4-ylacetyl-alanyl-isoglutaminyl-2,4-dinitrophenyllysyl-alanyl-arginine; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D009842:Oligopeptides; D009238:N-Acetylmuramoyl-L-alanine Amidase