Comparative Structure Analysis of the Multi-Domain, Cell Envelope Proteases of Lactic Acid Bacteria (original) (raw)
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Biodiversitas Journal of Biological Diversity
Hidayat H, Haryadi W, Raharjo TJ. 2020. Three-dimensional structure modeling of a protease from lactic acid bacteria Leuconostoc mesenteroides K7 using automated protein homology analysis. Biodiversitas 21: 3156-3162. This study aimed to characterize the protease encoding gene of Leuconostoc mesenteroides K7 isolated from Kelengkeng (Dimocarpus longan) fruit as well as to predict the structure of the protein using in silico approach. Gene characterization was performed using PCR employs primers designed based on protease gene of other Leuconostoc species, followed by cloning and sequencing of the PCR product. Protein structural modeling was targeted to the deduced amino acid sequence of the gene employ multiple sequence alignment and SWISS-Model online software. As a result, the sequence of the PCR product contains an open reading frame with a size of 1,140 bp, which can be translated into 379 amino acids. The amino acid sequence shares 98.60% identity with protease from Leuconostoc suionicum (AP017935.1). Three conserved sequences of QTDA, INPGNSGGPL, and FAIP are known as the signature from the Serine endoprotease DegS family are detected. The three-dimensional modeling structure application shows that the protein share similarity of 37.62% to Protease Do-like I chloroplastic that belong to serine protease.
Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis
Frontiers in Bioengineering and Biotechnology, 2020
The cell envelope proteinase (CEP) ofLactococcus lactisis a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains ofL. lactisare typically auxotrophic for several amino acids and in order to grow to high cell densities in milk they need an extracellular protease. The structure of the entire CEP enzyme is difficult to determine experimentally due to the large size and due to the attachment to the cell surface. We here describe the use of a combination of structure prediction tools to create a structural model for the entire CEP enzyme ofLactococcus lactis. The model has implications for how the bacterium interacts with casein micelles during growth in milk, and it has implications regarding the energetics of the proteolytic system. Our model for the CEP indicates that the catalytic triad is activated through a structural change caused by interaction with the substrate. The CEP ofL. lactismight become a useful model for the mode of action for en...
Journal of dairy science, 2015
Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Strepto...
Towards a proteomic map of Lactococcus lactis NCDO 763
Electrophoresis, 2000
Lactococcus lactis is a widely used bacteria in dairy industry, specially in cheese ripening. Numerous lactococcal enzymes and proteins are involved in this process. Proteomics makes it possible to deal with a high number of proteins and identify modification of their patterns in two-dimensional (2-D) gels. However, an annotated reference map is necessary prior to analyzing protein variations. We have begun to construct such a map in easily reproducible conditions and identify proteins.
Structural and kinetic determinants of protease substrates
Nature Structural & Molecular Biology, 2009
Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in -helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.
Molecular Microbiology, 2000
We identified an exported protease in Lactococcus lactis ssp. lactis strain IL1403 belonging to the HtrA/ DegP family. Inactivation of the chromosomal gene (htrA Ll) encoding this protease (HtrA Ll) results in growth thermo-sensitivity at very high temperatures (above 378C for L. lactis). The role of htrA Ll in extracellular proteolysis under normal growth conditions was examined by testing the stability of different exported proteins (i.e. fusions, a heterologous pre-pro-protein or a native protein containing repeats), having different locations. In the wild-type (wt) strain, degradation products, including the Cterminal protein ends, were present in the medium, indicating that proteolysis occurs during or after export to the cell surface; in one case, degradation was nearly total. In contrast, proteolysis was totally abolished in the htrA strain for all five proteins tested, and the yield of full-length products was significantly increased. These results suggest that HtrA Ll is the sole extracellular protease that degrades abnormal exported proteins. In addition, our results reveal that HtrA Ll is needed for the pro-peptide processing of a natural pro-protein and for maturation of a native protein. We propose that in lactococci, and possibly in other Gram-positive organisms with small sizedgenomes, a single surface protease, HtrA, is totally responsible for the housekeeping of exported proteins.
The proteotytic systems of lactic acid bacteria
Antonie van Leeuwenhoek, 1996
Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.
European Journal of Biochemistry, 1990
The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20: 1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1-214 and 21 5-300 are separated under protein-denaturing conditions. Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.
Scientific Reports, 2016
A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5 M −1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features. In the past decade, metagenomics has been utilized as a powerful technology for the discovery of novel enzymes and other valuable biomolecules produced by non-cultivated microbes 1,2. The majority of the research using this technology aims to demonstrate the distribution of genes in a specific environment. This includes the function assignment of putative proteins via sequence homology or activity-based assays 3,4. New enzymes have been isolated from metagenomic libraries constructed from various environments, many with potential for biotechnological and industrial applications 5–10. Amongst enzymes, amidases and peptidases/proteases are especially important in industry 11,12. A common substrate for these two groups of enzymes is the peptidoglycan present solely in bacterial cell walls 13 .