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

Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 (Lb. bulgaricus) is ch aracterized by a high level of peptidase activities specific to proline-con taining peptides. A prolidase (PepQ, EC 3.4.13.9) was purified to homogenei ty and characterized as a strict dipeptidase active on X-Pro dipeptides, ex cept Gly-Pro and Pro-Pro. The values for K-m and V-max were, respectively, 2.2 mM and 0.33 mmol min(-1) mg(-1), with Leu-Pro as the substrate. The enz yme exhibited optimal activity at 50 degrees C and ph 6.0. and required the presence of Zn2+. Sire exclusion chromatographies and SDS-PACE analysis le d to the conclusion that this prolidase was a homodimer. Antibodies raised against the purified protein allowed the detection of PepQ among several La ctobacillus species but not lactococci. The pepQ gene and the upstream regi on were isolated and sequenced. The deduced peptide sequence showed that Pe pQ belongs to the M24 family of metallopeptidases. The pepR1 gene is locate d immediately upstream of pepQ and its product is homologous to the transcr iption factor CcpA, which is involved in catabolite repression of catabolic operons from Cram-positive bacteria. The pepR1-pepQ intergenic region cont ains a consensus catabolite-responsive element (CRE) which could be a targe t for PepR1 protein. Moreover, in contrast to other proline-specific enzyme s from Lb. bulgaricus, PepQ biosynthesis was shown to be dependent on the c omposition of the culture medium, but not on the peptide concentration. A p ossible regulation mechanism is discussed.