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Cited by 40Enzyme Production and CharacterizationEnzyme Catalysis and ImmobilizationProbiotics and Fermented Foods

Engineering of the substrate-binding region of the sublilisin-like, cell-envelop proteinase of Lactococcus lactis

Siezen, Roland J., Bruinenberg, Paul G., Vos, Pieter, Alen-Boerrigter, Ingrid Van, Nijhuis, Monique, Alting, Arno C., Exterkate, Fred A., Vos, Willem M.de·1993·"Protein Engineering, Design and Selection"
DOI 10.1093/protein/6.8.927
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Abstract

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.

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Crossref · 10.1093/protein/6.8.927

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  • The AKT triplet could even be deleted or have insertions of 6 or 12 amino acids and still support growth with only minimal reduction in growth rate.

    “The AKT triplet could even be deleted or have insertions of 6 or 12 amino acids and still support growth with only minimal reduction in growth rate (Siezen et al., 1993).”

    From Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis (2020) · cites this paper as Siezen et al., 1993

  • Change of the serine of the catalytic triad to an alanine led to complete inactivation of the enzyme.

    “Change of the serine of the catalytic triad to an alanine led to complete inactivation of the enzyme.”

    From Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis (2020) · cites this paper as Siezen et al., 1993

  • In a third engineering study, positions AKT(137-139), N166, S433, and K748 corresponding to position 322-324, 351, 618, and 933 in MS22337 PrtP sequence; except for the serine of catalytic triad, all positions could be mutated without losing proteolytic activity.

    “These positions were AKT(137–139), N166, S433, and K748 corresponding to position 322–324, 351, 618, and 933 in the MS22337 PrtP sequence. Except for the serine, which is the serine of the catalytic triad, all the positions could be mutated without losing the proteolytic activity of the enzyme.”

    From Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis (2020) · cites this paper as Siezen et al., 1993

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Authors
Siezen, R. J., Bruinenberg, P. G., Vos, P., Alen-Boerrigter, I. V., Nijhuis, M., Alting, A. C., Exterkate, F. A., & Vos, W. M. D.
Affiliations
Nederlands Instituut Voor Zuivel Oonderzoek (NL)
ORCID
Venue
"Protein Engineering, Design and Selection"
Publisher
Oxford University Press (OUP)
Published
1993
Volume
6
Issue
8
Pages
927-937
ISSN
  • 0269-2139 (ISSN-L)
  • 1741-0126
  • 1741-0134
DOI
10.1093/protein/6.8.927
OpenAlex
W2164735085
Cited by
40
References
0
Citations by year
2025: 1 · 2023: 1 · 2020: 1 · 2012: 2
FWCI
3.93
Type
journal article
Language
en
Open access
closed
Locations
  • Protein Engineering Design and Selection (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
Keywords
Lactococcus lactis, cell envelope proteinase, subtilisin-like protease, substrate-binding region, site-directed mutagenesis, cassette mutagenesis, subtilisin, thermitase, substrate specificity, thermal stability, binding cleft, autoproteolysis, proteolytic activity
MeSH terms
Amino Acid Sequence, Animals, Bacterial Proteins, Base Sequence, Caseins, Catalysis, Cloning, Molecular, DNA Mutational Analysis, Enzyme Stability, Membrane Proteins, Milk, Models, Molecular, Molecular Sequence Data, Peptide Fragments, Serine Endopeptidases, Lactococcus lactis, Substrate Specificity, Protein Engineering, Mutagenesis, Site-Directed, Sequence Alignment

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