Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase

Abstract

Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (10 4 -fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available.Peptide-hydrolyzing enzymes are required for the complete degradation of proteins to free amino acids and for the utilization of peptides as nutrient sources. Salmonella enterica serovar Typhimurium contains at least 14 peptidases with various substrate specificities, ensuring that these two processes are carried out fully (12). Four of these peptidases (peptidases N, A, B, and D) have a broad substrate specificity, and previous studies have shown that a mutant lacking all four enzymes is unable to completely degrade intracellular proteins to free amino acids (19). These broad-specificity peptidases do not, however, hydrolyze all peptides equally well. For example, none of the four can hydrolyze X-Pro peptides (where X can be any amino acid). Specialized enzymes (peptidases P and Q) that specifically hydrolyze X-Pro peptides have evolved to carry out this function (11). Only one of the broad-specificity enzymes, peptidase B, is able to hydrolyze Asp-X peptides (Z. Mathew, T. M. Knox, and C. G. Miller, submitted for publication), and cells contain an additional enzyme, peptidase E, that is specific for Asp-X dipeptides (5).Peptidase E was first identified as an activity capable of hydrolyzing Asp-X peptides and was subsequently shown to have specificity for aspartyl dipeptides (5). It does not hydrolyze Glu-X, Asn-X, Gly-X, or any other nonaspartyl peptide that has been tested (5). Peptidase E requires a free N terminus and does not cleave N-blocked peptides. The only other Asp-X-specific peptidases ...

Dispensable genes and foreign DNA in Streptococcus mutans

“…1a). Ingbritt and LML7 contained an ORF that shows homology to the pepE, peptidase E, gene of Salmonella enterica that encodes a peptidase specific for aspartyl dipeptides (Larsen et al, 2001;Lassy & Miller, 2000) and has been named pepEH.…”

“…Peptidase assay. Extracts of S. mutans were prepared as described by Simpson & Russell (1998) and assayed for activity against Nacetyl-L-aspartic acid a-(p-nitroanilide) (Sigma) by measuring p-nitroaniline release at A 410 (Lassy & Miller, 2000).…”

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

“…Others have a very narrow range of activity. In S. enterica serovar Typhimurium, four well-characterized peptidases have been reported to be active in vitro toward N-terminal aspartyl peptides: PepB, a broad-specificity, leucine aminopeptidase family enzyme that hydrolyzes N-terminal aspartyl peptides of various lengths (24); PepE, an Asp-X-specific dipeptidase (8,10,14,20); IadA, an isoaspartyl dipeptidase (12, 19); and IaaA, an isoaspartyl aminopeptidase (19). The in vivo contribution of each of these peptidases to the utilization of N-terminal Asp peptides is unknown, but a strain containing null mutations in the genes encoding each of the N-terminal Asp-hydrolyzing peptidases can grow on Asp-Leu as a Leu source (19).…”

Biochemical and molecular characterization of a periplasmic hydrolase for oxidized polyvinyl alcohol from Sphingomonas sp. strain 113P3

“…PHB depolymerases share common structural domains conserved in the group as a whole: in the Nterminus, the signal peptide, the catalytic domain including the lipase box, the threonine-rich region or the type III module of fibronectin, and the substrate-binding site (Jendrossek et al, 1995a, b). The primary structure of the catalytic domain of these depolymerases contains certain conserved structures such as an oxyanion hole (histidine) and a triad of three amino acid residues (serine, aspartate and histidine) that is conserved among the serine proteases (Brenner, 1988;Kim et al, 2004;Lassy & Miller, 2000). Their consensus sequences are L***lHGC-QtAs, ID-n-vYV-GLS-G+--t, vw-G-sDyTV, and GM-H--P---G, respectively (* indicates hydrophobic, + a small side chain, and the corresponding residues are underlined).…”