title: Substrate-induced inactivation of the Escherichia coli AmiD N-acetylmuramoyl-L-alanine amidase highlights a new strategy to inhibit this class of enzyme.
abstract: In the eubacterial cell, the peptidoglycan is perpetually hydrolyzed throughout the cell cycle by different enzymes such as lytic transglycosylases, endopeptidases, and amidases. In Escherichia coli, four N-acetylmuramoyl-l-alanine amidases, AmiA, -B, -C, and -D, are present in the periplasm. AmiA, -B, and -C are soluble enzymes, whereas AmiD is a lipoprotein anchored in the outer membrane. To determine more precisely the specificity and the kinetic parameters of AmiD, we overproduced and purified the native His-tagged AmiD in the presence of detergent and a soluble truncated form of this enzyme by removing its signal peptide and the cysteine residue responsible for its lipidic anchorage. AmiD is a zinc metalloenzyme and is inactivated by a metal chelator such as EDTA. Native His-tagged and truncated AmiD hydrolyzes peptidoglycan fragments that have at least three amino acids in their peptide chains, and the presence of an anhydro function on the N-acetylmuramic acid is not essential for its activity. The soluble truncated AmiD exhibits a biphasic kinetic time course that can be explained by the inactivation of the enzyme by the substrate. This behavior highlights a new strategy to inhibit this class of enzymes.
authors.affiliation: Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart Tilman, Allée de la Chimie 6, 4000 Liège, Belgium.
authors.forename: Anne
authors.initials: A
authors.lastname: Pennartz
authors.forename: Catherine
authors.initials: C
authors.lastname: Généreux
authors.forename: Claudine
authors.initials: C
authors.lastname: Parquet
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
authors.forename: Bernard
authors.initials: B
authors.lastname: Joris
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D010457:Peptidoglycan; D009238:N-Acetylmuramoyl-L-alanine Amidase