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

Microbiology (Reading). 1999 Feb;145 ( Pt 2):437-446. doi: 10.1099/13500872-145-2-437.

Abstract

Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 (Lb. bulgaricus) is characterized by a high level of peptidase activities specific to proline-containing peptides. A prolidase (PepQ, EC 3.4.13.9) was purified to homogeneity and characterized as a strict dipeptidase active on X-Pro dipeptides, except Gly-Pro and Pro-Pro. The values for Km and Vmax were, respectively, 2.2 mM and 0.33 mmol min(-1) mg(-1), with Leu-Pro as the substrate. The enzyme exhibited optimal activity at 50 degrees C and pH 6.0, and required the presence of Zn2+. Size exclusion chromatographies and SDS-PAGE analysis led to the conclusion that this prolidase was a homodimer. Antibodies raised against the purified protein allowed the detection of PepQ among several Lactobacillus species but not lactococci. The pepQ gene and the upstream region were isolated and sequenced. The deduced peptide sequence showed that PepQ belongs to the M24 family of metallopeptidases. The pepR1 gene is located immediately upstream of pepQ and its product is homologous to the transcription factor CcpA, which is involved in catabolite repression of catabolic operons from Gram-positive bacteria. The pepR1-pepQ intergenic region contains a consensus catabolite-responsive element (CRE) which could be a target for PepR1 protein. Moreover, in contrast to other proline-specific enzymes from Lb. bulgaricus, PepQ biosynthesis was shown to be dependent on the composition of the culture medium, but not on the peptide concentration. A possible regulation mechanism is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Chromosome Mapping
  • Dipeptidases / biosynthesis
  • Dipeptidases / chemistry*
  • Dipeptidases / genetics
  • Dipeptidases / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial
  • Gene Expression Regulation, Enzymologic
  • Humans
  • Lactobacillus / enzymology*
  • Lactobacillus / genetics
  • Lactobacillus / growth & development
  • Lactococcus / enzymology
  • Molecular Sequence Data
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Substrate Specificity

Substances

  • Dipeptidases
  • proline dipeptidase

Associated data

  • GENBANK/Y13385