Deletion of Various Carboxy-Terminal Domains of Lactococcus lactis SK11 Proteinase: Effects on Activity, Specificity, and Stability of the Truncated Enzyme
Open accessAbstract
The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.
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At least part of the B domain of PrtB is involved in stabilization of the catalytic domain.
“This suggests that at least a part of the B domain is involved in stabilization of the catalytic domain of PrtB, similar to the situation described with lactococcal PrtP (3).”
Bruinenberg et al. (2000) concluded that domains B, H, and W are dispensable for protease activity and have no role in determining the specificity of the CEP enzyme.
“The role of domains B, H, and W in determining the specificity of CEP has been investigated by Bruinenberg et al. (2000), and they concluded that these domains are dispensable for protease activity and that they have no role in determining the specificity of the CEP enzyme.”
From Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis (2020) · cites this paper as Bruinenberg et al., 2000
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Germond, J.-E., Delley, M., Gilbert, C., & Atlan, D. (2003) · Applied and Environmental Microbiology · doi:10.1128/aem.69.6.3377-3384.2003 · Cited by 36 · see citing works
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Lactocepin: the cell envelope-associated Endopeptidase of lactococci.
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- Authors
- Bruinenberg, P. G., De Vos, W. M., & Siezen, R. J.
- Affiliations
- NIZO food research, Ede, The Netherlands
- Nederlands Instituut Voor Zuivel Oonderzoek (NL)
- ORCID
- Siezen, Roland J.
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2000-07
- Volume
- 66
- Issue
- 7
- Pages
- 2859-2865
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC92084
- OpenAlex
- W2171585397
- Cited by
- 13
- References
- 61
- Citations by year
- 2024: 1 · 2023: 1 · 2020: 1 · 2014: 1 · 2012: 2
- FWCI
- 2.83
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Applied and Environmental Microbiology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Socio-Environmental Systems Modeling (acceptedVersion)
- Radboud Repository (Radboud University) (submittedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- Funders
- European Commission (BIOT-CT91-0263)
- Rijksuniversiteit Groningen
- Topics
- Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Keywords
- cell envelope proteinase, C-terminal domain, B domain, serine protease, autoproteolysis, enzyme stability, proteolytic specificity, deletion mutants, monoclonal antibody epitopes, lactic acid bacteria
- MeSH terms
- Caseins, Endopeptidases, Lactococcus lactis, Structure-Activity Relationship, Substrate Specificity, Mutagenesis, Sequence Deletion, Protein Structure, Tertiary, Epitope Mapping
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