Substrate-induced inactivation of the Escherichia coli AmiD N-acetylmuramoyl-L-alanine amidase highlights a new strategy to inhibit this class of enzyme.
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Pennartz, Anne
Genereux, Catherine ; Université de Liège - ULiège > Département des sciences cliniques > Labo de biologie des tumeurs et du développement
Parquet, Claudine
Mengin-Lecreulx, Dominique
Joris, Bernard ; Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes - Centre d'ingénierie des protéines
Language :
English
Title :
Substrate-induced inactivation of the Escherichia coli AmiD N-acetylmuramoyl-L-alanine amidase highlights a new strategy to inhibit this class of enzyme.
Publication date :
2009
Journal title :
Antimicrobial Agents and Chemotherapy
ISSN :
0066-4804
eISSN :
1098-6596
Publisher :
American Society for Microbiology (ASM), Washington, United States - District of Columbia
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