PREVENTION OF C-TERMINAL AUTOPROCESSING OF LACTOCOCCUS-LACTIS SK11 CELL-ENVELOPE PROTEINASE BY ENGINEERING OF AN ESSENTIAL SURFACE LOOP

The catalytic domain of the cell-envelope proteinase from Lactococcus lactis SK11 has Various inserts, situated in external loops of the cat alytic domain, compared with the related subtilisins. Protein engineer ing was employed to analyse the necessity and function of one of these extra loops (residues 205-219), that is predicted to be located in cl ose proximity to the substrate-binding region and is susceptible to au toproteolysis. We constructed a deletion mutant which lacks 14 residue s of this surface loop and subsequently introduced various insertion c assettes coding either for the original loop with three mutations (E20 5S/E218T/M219S: triple-mutant proteinase) or for neutral spacers (1, 4 , 7 and 16 serine residues). Engineered proteinases were analysed for activity, (auto)processing, and cleavage specificity. The presence of residues 205-219 is shown to be essential for proteolytic activity, as only triple-mutant proteinase retained activity towards casein substr ates. The triple-mutant proteinase was found to be defective in C-term inal autoprocessing, and subsequent release from the lactococcal cell envelope in a calcium-free medium, indicative of either an altered pro teolytic specificity or altered accessibility of the processing site. The specificity change appears to be subtle, as only small differences were found between wild-type and triple-mutant proteinase in the brea kdown of casein substrates.