CLONING AND NUCLEOTIDE-SEQUENCE OF THE CYCLIC-AMP RECEPTOR PROTEIN-REGULATED SALMONELLA-TYPHIMURIUM PEPE GENE AND CRYSTALLIZATION OF ITS PRODUCT, AN ALPHA-ASPARTYL DIPEPTIDASE

The Salmonella typhimurium pepE gene, encoding an N-terminal-Asp-speci fic dipeptidase, has been cloned on pBR328 by complementation of the A sp-Pro growth defect conferred by a pepE mutation. Strains carrying th e complementing plasmids greatly overproduce peptidase E. The enzyme h as been purified from an extract of such a strain, its N-terminal amin o acid sequence has been determined, and crystals suitable for X-ray d iffraction have been grown. A new assay using L-aspartic acid p-nitroa nilide as a substrate has been used to determine the pH optimum (appro ximate to 7.5) and to test the effect of potential inhibitors. Inserti ons of transposon gamma delta (Tn1000) into one of the plasmids have b een used to localize the gene and as sites for priming sequencing reac tions. The nucleotide sequence of a 1,088-bp region of one of these pl asmids has been determined. This sequence contains an open reading fra me that predicts a 24.8-kDa protein with an N-terminal sequence that a grees 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 p romoter 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 f usion indicate that pepE is a member of the CRP regulon. The unique sp ecificity of peptidase E and its lack of sequence similarity to any ot her peptidase suggest that this enzyme may be the prototype of a new c lass of peptidases. Its regulation by CRP and its specificity suggest that the enzyme may play a role in allowing the cell to use peptide as partate to spare carbon otherwise required for the synthesis of the as partate family of amino acids.