Evidence for a large dispensable segment in the subtilisin-like catalytic domain of the Lactococcus lactis cell-envelope proteinase.
The Lactococcus lactis SK11 cell-envelope proteinase contains various inserts, located in external loops of the catalytic domain compared with related subtilisins. In this study, protein engineering was employed to determine the function of the largest loop insertion (residues 238-388) relative to the subtilisin structure. By site-directed mutagenesis we have deleted the fragment of the proteinase gene encoding these 151 residues and analyzed the mutant delta 238-388 proteinase for activity, (auto)processing and cleavage specificity. This extra segment is found to be inessential for activity, and its removal does not inhibit folding as the mutant proteinase is still active. In addition, the N- and C-terminal autoprocessing of the delta 238-388 proteinase appears to be unchanged. However, removal of residues 238-388 altered substantially the caseinolytic specificity of the enzyme, indicating that this extra segment influences substrate specificity. Residues 238-388 were shown to contain a specific epitope for a monoclonal antibody.
Bruinenberg
P G
PG
Department of Biophysical Chemistry, NIZO, Ede, The Netherlands.
Doesburg
P
P
Alting
A C
AC
Exterkate
F A
FA
de Vos
W M
WM
Siezen
R J
RJ
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002473:Cell Wall / Q000201:enzymology*; D003001:Cloning, Molecular; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D012697:Serine Endopeptidases; D013381:Subtilisins; C085783:lactocepin