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

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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.

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