Substrate-induced inactivation of the Escherichia coli AmiD N-acetylmuramoyl-L-alanine amidase highlights a new strategy to inhibit this class of enzyme.
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.
Pennartz
Anne
A
Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart Tilman, Allée de la Chimie 6, 4000 Liège, Belgium.
Généreux
Catherine
C
Parquet
Claudine
C
Mengin-Lecreulx
Dominique
D
Joris
Bernard
B
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
D029968:Escherichia coli Proteins; D010457:Peptidoglycan; D009238:N-Acetylmuramoyl-L-alanine Amidase