Files in this item

FilesDescriptionFormat

application/pdf

application/pdf3131065.pdf (7MB)Restricted to U of Illinois
(no description provided)PDF

Description

Title:Hydrolysis and Toxicity of Aspartyl Peptides and the Influence of Metal Ions on Aspartyl Peptide Hydrolases in Salmonella Enterica Serovar Typhimurium
Author(s):Broder, Daniel Howard
Doctoral Committee Chair(s):Miller, Charles G.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Chemistry, Biochemistry
Abstract:Extracts of a multiply peptidase deficient (MPD)(pepN,A,B,D,P,Q,T,E,iadA,iaaA ) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity which 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. Disruption of the dapE gene and transfer of the mutation into a MPD strain yielded a strain with extract inactive towards Asp-Leu or any other peptide tested. This strain would not utilize any peptides tested as amino acid sources. Purified DapE also exhibited Mn2+-dependent activity towards aspartyl peptides and specificity studies with pure enzyme revealed it was specific for aspartyl dipeptides. Metal binding experiments indicated that, of two metal binding sites of unequal affinity in DapE, Mn 2+ is bound to the low affinity site and Zn2+ to the high affinity site when DapE functions as a peptidase. Overexpression of DapE in the MPD dapE strain conferred strong growth on Asp-Leu as a Leu source indicating DapE functions well as a peptidase when overexpressed. Aspartyl peptides were demonstrated to be toxic to the MPD dapE strain, but not to a wild type peptidase strain. This bacteriocidal effect could be antagonized by reintroduction of peptidases specific for aspartyl peptides. Growth of MPD dapE in the absence of aspartyl peptides was very slow, but could be enhanced by the introduction of aspartyl peptidases suggesting that internally generated toxic aspartyl peptides cause slow growth of peptidase deficient strains. Overproduction of aspartyl dipeptide specific peptidases nearly completely rescued slow growth of MPD dapE. Transduction of MPD dapE and selection for growth on Asp-Leu as a Leu source revealed pepD, pepA, pepE, and pepB . Other strains containing a single peptidase gene were constructed. Extracts from the pepA+, pepB +, pepD+, and dapE + strains all showed activation by Mn2+ for aspartyl peptidase activity. Disruption of Mn2+ transport dramatically affected aspartyl peptide utilization by pepA+, pepD+, and dapE+-strains, indicating that these enzymes all utilize Mn2+ in vivo for aspartyl peptidase activity. The implications of Mn2+ activation of peptidases and the potential variability of peptidase bound cations in vivo are discussed.
Issue Date:2003
Type:Text
Language:English
Description:126 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2003.
URI:http://hdl.handle.net/2142/86668
Other Identifier(s):(MiAaPQ)AAI3131065
Date Available in IDEALS:2015-09-28
Date Deposited:2003


This item appears in the following Collection(s)

Item Statistics