GENE CLONING AND CHARACTERIZATION OF PEPC, A CYSTEINE AMINOPEPTIDASE FROM STREPTOCOCCUS-THERMOPHILUS, WITH SEQUENCE SIMILARITY TO THE EUKARYOTIC BLEOMYCIN HYDROLASE

Streptococcus thermophilus CNRZ 302 contains at least three general am inopeptidases able to hydrolyze Phe-beta-naphthylamide substrate. The gene encoding one of these aminopeptidases was cloned from a total DNA library of S. thermophilus CNRZ 302 constructed in Escherichia coli T G1 using pBluescript plasmid. The wild-type TG1 strain, although not d eficient in aminopeptidase activity, is unable to hydrolyze the substr ate Phe-beta-naphthylamide, and thus the library could be screened wit h an enzymic plate assay using this substrate. One clone was selected which was shown to express an aminopeptidase, identified as a PepC-lik e enzyme on the basis of cross-reactivity with polyclonal antibodies d irected against the lactococcal PepC cysteine aminopeptidase. The gene was further subcloned and sequenced. A complete open reading frame co ding for a 445-residue (50414 Da) polypeptide was identified. 70% iden tity was found between the deduced amino acid sequence and the sequenc e of PepC from Lactococcus lactis subspecies cremoris, confirming the identity of the cloned gene. High sequence similarity (38% identity) w as also found with an eucaryotic enzyme, bleomycin hydrolase. Ln addit ion, the predicted amino acid sequence of the streptococcal PepC showe d a region of strong similarity to the active site of cysteine protein ases with conservation of the residues involved in the catalytic site. The product of the cloned pepC gene was overproduced in E. coli and w as purified from a cellular extract. Purification to homogeneity was a chieved by two-step ion-exchange chromatography. Biochemical character ization of the pure recombinant enzyme confirms that the cloned peptid ase is a thiol aminopeptidase possessing a broad specificity. The enzy me has a molecular mass of 300 kDa suggesting an hexameric structure. On the basis of sequence similarities as well as common biochemical an d enzymic properties, the bacterial PepC-type enzymes and the eucaryot ic bleomycin hydrolase constitute a new family of thiol aminopeptidase s among the cysteine peptidases.