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Journal of Bacteriology, August 2003, p. 4748-4754, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4748-4754.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

DapE Can Function as an Aspartyl Peptidase in the Presence of Mn²⁺

Daniel H. Broder and Charles G. Miller^*

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 10 April 2003/ Accepted 29 May 2003

Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn²⁺. Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn²⁺-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-His₆). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn²⁺-dependent aspartyl peptidase activity relative to the MPD dapE⁺ strain. In addition, purified DapE-His₆ exhibited Mn²⁺-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 Zn²⁺ in the high-affinity site and Mn²⁺ in the low-affinity site.

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* Corresponding author. Mailing address: Department of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemical and Life Science Laboratory, 601 South Goodwin Ave., Urbana, IL 61801. Phone: (217) 244-8418. Fax: (217) 244-6697. E-mail: charlesm{at}uiuc.edu.

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Journal of Bacteriology, August 2003, p. 4748-4754, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4748-4754.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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