oa Screening for and cloning and molecular characterization of two new oligopeptidase B encoding genes

Oligopeptidase B (opdB, EC 3.4.21.83) is a member of the prolyl oligopeptidase family of serine peptidases and unrelated to the trypsin and subtilisin families. It is a potential processing enzyme of prokaryotes to produce biologically active products, being very specific for the basic amino acid pairs of polypeptides. Bacterial oligopeptidase B cleaves globular proteins, albeit in a highly restricted fashion. While most members of this peptidase family hydrolyse peptide bonds at the C-terminal side of proline residues, oligopeptidase B exhibits a trypsin-like substrate specificity, cleaving peptides after basic residues (arginine or lysine). Oligopeptidase B was first cloned and characterized from Escherichia coli, and has also been described in other prokaryotes. Similar enzymes have been found in plants and some other higher organisms. We report the isolation of two different new oligopeptidase B bacterial strains producers. We identified the two genes, designated opdB1 and opB2. The opdB genes encodes a 703-residue peptide with high homology to the oligopeptidase B family in prokaryotes. The isolated opdBs gave the highest similarity score to oligopeptidase B of Stenotrophomonas maltophilia strain K279a (GenBank AM743169). To reveal the structural and kinetic properties of oligopeptidase B in more detail, we have cloned, expressed, and purified the enzymes to produce sufficient material to help in physical investigations, including NMR and x-ray crystallographic measurements. We also carried out a molecular characterization study of the two enzymes.