Studying a cell division amidase using defined peptidoglycan substrates.
Three periplasmic N-acetylmuramoyl-l-alanine amidases are critical for hydrolysis of septal peptidoglycan, which enables cell separation. The amidases cleave the amide bond between the lactyl group of muramic acid and the amino group of l-alanine to release a peptide moiety. Cell division amidases remain largely uncharacterized because substrates suitable for studying them have not been available. Here we have used synthetic peptidoglycan fragments of defined composition to characterize the catalytic activity and substrate specificity of the important Escherichia coli cell division amidase AmiA. We show that AmiA is a zinc metalloprotease that requires at least a tetrasaccharide glycopeptide substrate for cleavage. The approach outlined here can be applied to many other cell wall hydrolases and should enable more detailed studies of accessory proteins proposed to regulate amidase activity in cells.
Lupoli
Tania J
TJ
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Taniguchi
Tohru
T
Wang
Tsung-Shing
TS
Perlstein
Deborah L
DL
Walker
Suzanne
S
Kahne
Daniel E
DE
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002455:Cell Division; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology; D029968:Escherichia coli Proteins; D008666:Metalloendopeptidases; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D015032:Zinc
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D010457:Peptidoglycan; D008666:Metalloendopeptidases; D000581:Amidohydrolases; C019665:amidase; D015032:Zinc