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Journal of Bacteriology, June 2002, p. 3329-3337, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3329-3337.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Substrate Recognition Properties of Oligopeptidase B from Salmonella enterica Serovar Typhimurium

Rory E. Morty,1,{dagger} Vilmos Fülöp,2 and Norma W. Andrews1*

Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536,1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom2

Received 23 January 2002/ Accepted 26 March 2002

Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P1. While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu576 and Glu578, that define P1 specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp460 and Asp462, that may be involved in defining P2 specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.

* Corresponding author. Mailing address: Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06536. Phone: (203) 737-2410. Fax: (203) 737-2630. E-mail: norma.andrews{at}yale.edu.

{dagger} Present address: Zentrum für Innere Medizin, Medizinische Klinik II, Justus Liebig Universität, D-35392 Gießen, Germany.

Journal of Bacteriology, June 2002, p. 3329-3337, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3329-3337.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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