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, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal 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 lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.
Bruinenberg
P G
PG
NIZO food research, Ede, The Netherlands.
De Vos
W M
WM
Siezen
R J
RJ
D002364:Caseins / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018604:Epitope Mapping; D013294:Lactococcus lactis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002364:Caseins; D010450:Endopeptidases