Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 proteinase: Effects on activity, specificity, and stability of the truncated enzyme

The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, m ultidomain protein of nearly 2,000 residues consisting of an N-terminal ser ine protease domain, followed by various other domains of largely unknown f unction, Using a strategy of deletion mutagenesis, we have analyzed the fun ction of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various de letion mutants were functionally expressed in L. lactis and analyzed for en zyme stability, activity, (auto)processing, and specificity toward several substrate. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secre ted proteinases of unaltered specificity. Gradually increasing the C-termin al deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lac king the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. T his suggests that the main role played by these domains is providing stabil ity or protection from autoproteolysis (B domain), spacing away from the ce ll (H domain), and anchoring to the cell envelope (W and AN domains). In ad dition, this study allowed us to more precisely map the main C-terminal aut oprocessing site of the SK11 proteinase and the epitope for binding of grou p IV monoclonal antibodies.