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 catalytic domain, compared with the related subtilisins. Protein engineering was employed to analyse the necessity and function of one of these extra loops (residues 205-219), that is predicted to be located in close proximity to the substrate-binding region and is susceptible to autoproteolysis. We constructed a deletion mutant which lacks 14 residues of this surface loop and subsequently introduced various insertion cassettes coding either for the original loop with three mutations (E205S/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 substrates. The triple-mutant proteinase was found to be defective in C-terminal autoprocessing, and subsequent release from the lactococcal cell envelope in a calcium-free medium, indicative of either an altered proteolytic 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 breakdown of casein substrates.
Bruinenberg
P G
PG
Department of Biophysical Chemistry, NIZO, Ede, The Netherlands.
de Vos
W M
WM
Siezen
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008892:Milk / Q000382:microbiology; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C085783:lactocepin