Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis

Abstract

The cell envelope proteinase (CEP) of Lactococcus lactis is a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains of L. lactis are 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 of Lactococcus 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 of L. lactis might become a useful model for the mode of action for enzymes belonging to the large class of S8 proteinases with a PA (protease associated) domain and a downstream fibronectin like domain.

Comparative Structure Analysis of the Multi-Domain, Cell Envelope Proteases of Lactic Acid Bacteria

“…The structural fold of the N-terminal region was similar to the H-domain in PrtH (RMSD around 2.8 Å), whereas the C-terminal region was larger, with five longer helices. In the structure models, the C-terminal region had two different orientations, indicating a potential flexibility of the H-domain ( Figure 7 D) with the possibility to adopt more compact structures, as previously predicted [ 21 ]. The structural similarities in the H-domains suggested that these H-domains had similar functions.…”

“…Fn2 β4–β5 orientated towards the active site with approximately 15 Å from the catalytic triad when Fn2 β4–β5 formed an extended β-turn structure or a β-hairpin. This region had also been proposed to be involved in substrate specificity [ 21 ]. However, Fn2 β4–β5 was much shorter in PrtP NFICC96H than in the other PrtP homologs, locating Fn2 β4–β5 further away from the catalytic cleft.…”

“…The propeptide is not the only required factor for assisting the maturation process of PrtP homologs, some of which require the prolyl cis / trans isomerase activity of the protein folding chaperone PrtM to achieve their active conformations [ 51 ]. The proline residues required for these proteases’ conformational changes have not been identified, but critical proline residues have been proposed to be located in the PR-domains as the autocatalytic processing can depend on the absence of PrtM [ 21 , 51 ]. However, the PrtP homologs may follow different maturation steps, as not all PrtP homologs seem to depend on PrtM maturation [ 8 , 13 , 15 ], and ScpC is cleaved in its PR1, working as a heterodimer ( Figure 2 ) [ 17 , 20 ].…”

“…Homology modeling has provided structural insight into the Lactococcus lactis PrtP homologs, using subtilisin and ScpA structures as templates [ 5 , 10 ]. A combined approach using different homology modeling tools achieved the first three-dimensional model of an entire L. lactis PrtP, which is from L. lactis MS22337 derived from spontaneously acidified camel milk [ 21 ]. This structural model implicates the bacterial interaction with casein micelles.…”

Fermentation of plant‐based dairy alternatives by lactic acid bacteria

“…These are secreted from the cell and anchored to the cell membrane, which ensures that proteolysis occurs close to the cell and allows efficient subsequent transportation. The catalytic domain contains the active site, composed of three amino acid residues (serine, histidine and aspartic acid) that are highly conserved across species (Hansen and Marcatili, 2020 ). The cell envelope proteinase from Lc.…”