DapE can function as an aspartyl peptidase in the presence of Mn2+.
Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn(2+). Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn(2+)-dependent peptidase is DapE (N-succinyl-L,L-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His(6)). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn(2+)-dependent aspartyl peptidase activity relative to the MPD dapE(+) strain. In addition, purified DapE-His(6) exhibited Mn(2+)-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn(2+) in the high-affinity site and Mn(2+) in the low-affinity site.
Broder
Daniel H
DH
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Miller
Charles G
CG
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D000581:Amidohydrolases; D016282:Aspartic Acid Endopeptidases; D008345:Manganese; D011994:Recombinant Proteins; D015032:Zinc; C054685:N-succinyl-l,l-diaminopimelic acid desuccinylase