Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis.
The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase. Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23). Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged. The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site. Several mutant SK11 proteinases have novel properties not previously encountered in natural variants. Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity. Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase. Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.
Siezen
R J
RJ
Department of Biophysical Chemistry, NIZO, Ede, The Netherlands.
Bruinenberg
P G
PG
Vos
P
P
van Alen-Boerrigter
I
I
Nijhuis
M
M
Alting
A C
AC
Exterkate
F A
FA
de Vos
W M
WM
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008892:Milk / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D015202:Protein Engineering; D016415:Sequence Alignment; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002364:Caseins; D008565:Membrane Proteins; D010446:Peptide Fragments; D012697:Serine Endopeptidases; C085783:lactocepin