Characterization of a streptococcal endopeptidase with homology to human endothelin-converting enzyme

A gene encoding an endopeptidase from Streptococcus parasanguis FW213 has b een cloned and shown to have high sequence homology to genes encoding mamma lian metalloendopeptidases. The gene, designated S. parasanguis pepO, was c loned into the pET28a expression vector, resulting in a fusion of vector se quences encoding a hexahistidine tag at the carboxyl terminus. The recombin ant PepO (rPepO) was expressed in Escherichia coli and purified using an Ni 2+ affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and imm unoblot analysis revealed that other oral streptococci contain regions of D NA with homology to pepO and produce a protein with antigenic properties si milar to that of FW213 PepO. Enzymatic activity assays indicated that only S. parasanguis species possess the ability to cleave metenkephalin, the nat ural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that S. parasanguis PepO is a member of the M13 category of metall oendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE -1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that S. parasanguis PepO is more similar to ECE-1 t han to NEP.