DapE Can Function as an Aspartyl Peptidase in the Presence of Mn ²⁺

Abstract

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.Salmonella enterica serovar Typhimurium contains at least 12 peptidases able to degrade intracellular peptides (25). These peptidases vary in their substrate specificity. Some show a very broad substrate specificity hydrolyzing peptides of various lengths and amino acid compositions. Others have a very narrow range of activity. In S. enterica serovar Typhimurium, four well-characterized peptidases have been reported to be active in vitro toward N-terminal aspartyl peptides: PepB, a broad-specificity, leucine aminopeptidase family enzyme that hydrolyzes N-terminal aspartyl peptides of various lengths (24); PepE, an Asp-X-specific dipeptidase (8,10,14,20); IadA, an isoaspartyl dipeptidase (12, 19); and IaaA, an isoaspartyl aminopeptidase (19). The in vivo contribution of each of these peptidases to the utilization of N-terminal Asp peptides is unknown, but a strain containing null mutations in the genes encoding each of the N-terminal Asp-hydrolyzing peptidases can grow on Asp-Leu as a Leu source (19). An additional Asp-Leu-hydrolyzing activity was identified in extracts of this strain, but this activity has not been characterized. Peptidase activity in these extracts was only detectable in the presence of manganese. The goal of this work was to identify this Mn 2ϩ -dependent peptidase and the gene encoding it and to characterize its...

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