DCP GENE OF ESCHERICHIA-COLI - CLONING, SEQUENCING, TRANSCRIPT MAPPING, AND CHARACTERIZATION OF THE GENE-PRODUCT

Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichi a coli. We have isolated the respective gene, dcp, from a low-copy-num ber plasmid library by its ability to complement a dcp mutation preven ting the utilization of the unique substrate N-benzoyl-L-glycyl-L-hist idyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecula r mass determination of the purified dcp product. Transcript mapping b y primer extension and S1 protection experiments verified the physiolo gical significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expr ession. The predicted amino acid sequence of dipeptidyl carboxypeptida se, containing a potential zinc-binding site, is highly homologous (78 .8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium op dA and the E. coli prlC genes and to some metalloproteases from rats a nd Saccharomyces cerevisiae. No potential export signals could be infe rred from the amino acid sequence. Dipeptidyl carboxypeptidase was enr iched 80-fold from crude extracts of E. coli and used to investigate s ome of its biochemical and biophysical properties.