Cloning and nucleotide sequence of the cyclic AMP receptor protein-regulated Salmonella typhimurium pepE gene and crystallization of its product, an alpha-aspartyl dipeptidase

Abstract

The Salmonella typhimurium pepE gene, encoding an N-terminal-Asp-specific dipeptidase, has been cloned on pBR328 by complementation of the Asp-Pro growth defect conferred by a pepE mutation. Strains carrying the complementing plasmids greatly overproduce peptidase E. The enzyme has been purified from an extract of such a strain, its N-terminal amino acid sequence has been determined, and crystals suitable for X-ray diffraction have been grown. A new assay using L-aspartic acid p-nitroanilide as a substrate has been used to determine the pH optimum (-7.5) and to test the elfect of potential inhibitors. Insertions of transposon y8 (TnlOOO) into one of the plasmids have been used to localize the gene and as sites for priming sequencing reactions. The nucleotide sequence of a 1,088-bp region of one of these plasmids has been determined. This sequence contains an open reading frame that predicts a 24.8-kDa protein with an N-terminal sequence that agrees with that determined for peptidase E. The predicted peptidase E amino acid sequence is not similar to that of any other known protein. The nucleotide sequence of the region upstream from pepE contains a promoter with a cyclic AMP receptor protein (CRP) site, and the effects of growth medium and of a crp mutation on expression of a pepE-lacZ fusion indicate that pepE is a member of the CRP regulon. The unique specificity of peptidase E and its lack of sequence similarity to any other peptidase suggest that this enzyme may be the prototype of a new class of peptidases. Its regulation by CRP and its specificity suggest that the enzyme may play a role in allowing the cell to use peptide aspartate to spare carbon otherwise required for the synthesis of the aspartate family of amino acids.

Characterization of a prolidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 with an unusual regulation of biosynthesis

“…The biosynthetic regulation of two peptidases by components implicated in catabolite repression has already been described in Salmonella typhimurium (Conlin et al, 1994;Lombard0 et al, 1997). The physiological significance of such a mechanism is not known.…”

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

“…This study Assays of peptidase E activity. Peptidase E activity was measured by determining the rate of Asp-pNA hydrolysis (6). The reaction mixture contained either purified enzyme or crude cell extract in 0.1 M Tris-Cl (pH 7.5) and 1 mM Asp-pNA (from a 50 mM stock solution in dimethylformamide).…”

“…The selection primer, PstI/XhoI, was used with each of the mutagenic primers, all of which are listed in Table 2. A pepE plasmid, pCM247 [pBluescript II KS(ϩ) with a 2.6-kb serovar Typhimurium genomic DNA insert containing pepE and two adjacent genes], was used as the template (6). Mutated plasmids were screened for loss of a PstI site and gain of an XhoI site by corresponding restriction endonuclease digestion.…”

“…Peptidase E is not sensitive to inhibitors of the major classes of peptidases (6), and its sequence does not clearly place it in any known class of enzymes (13). No structural motifs that could identify peptidase E as a member of a known family have been recognized.…”

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

“…Aspartyl dipeptidase is not inactivated by the conventional serine protease inhibitor DFP or PMSF (phenyl methane sulfonyl fluoride) (Conlin et al 1994). While initial site-directed mutagenesis studies suggested that aspartyl dipeptidase was a classical serine protease, the structure of aspartyl dipeptidase from Salmonella typhimurium revealed that it in fact belongs to a new serine peptidase family with a Ser/His/Glu triad (Hakansson et al 2000).…”