A Characterization of Aspartyl Peptidases in Salmonella Typhimurium
Lassy, Rachel Anne Larsen
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https://hdl.handle.net/2142/86755
Description
Title
A Characterization of Aspartyl Peptidases in Salmonella Typhimurium
Author(s)
Lassy, Rachel Anne Larsen
Issue Date
2000
Doctoral Committee Chair(s)
Miller, Charles G.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Few of the peptidases previously characterized from Salmonella typhimurium are capable of hydrolyzing aspartyl peptides. Peptidase E, an aspartyl specific dipeptidase, was the first member of a new family of peptide hydrolases that now includes peptidases from other proteobacteria and from two eukaryotes. It is shown that Peptidase E from Xenopus laevis has the same substrate specificity as Peptidase E from S. typhimurium, indicating that in this family of enzymes both the catalytic and the substrate specificity have been conserved. An alignment of the amino acid sequences of the Peptidase E family allowed for potentially important residues to be identified. Through site-directed mutagenesis of the S. typhimurium pepE, it was proposed that this enzyme is a serine hydrolase that utilizes a catalytic triad of serine, aspartate, and histidine. In extracts of a pepE strain, two additional Asp-X hydrolases were detected. The identity of each of these enzymes as well as evidence of a third Asp-X hydrolase are presented in this thesis. Two of these enzymes are shown most rapidly hydrolyze isoaspartyl peptides (also called beta-aspartyl peptides). Of the two isoaspartyl peptidases, one is a homolog of IadA from E. coli and the other is described for the first time in this work and is encoded by a previously unknown open reading frame called ybiK in E. coli. The ybiK gene product is a threonine hydrolase that is a member of the Ntn (N-terminal nucleophile) family of enzymes. It is synthesized as a 32 kD polypeptide that dimerizes and undergoes processing, presumably autocatalytic, into an active form, which is a 60 kD heterotetramer. A strain of S. typhimurium lacking all of the broad specificity peptidases, as well as the aspartyl peptidases described here is still capable of growth on Asp-Leu as a leucine source. This growth is attributed to the remaining peptidase. This peptidase is specific for alpha-aspartyl peptides, has a native molecular mass of 70 kD, and has the unusual property of requiring manganese for activity.
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