Abstract
Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.
Highlights
Lanthipeptides are posttranslationally modified peptides that contain dehydrated amino acids andlanthionine residues (Knerr and van der Donk, 2012; Arnison et al, 2013)
The presence of an appropriate protease in the mix will release active nisin, we can monitor the activity of NisP by measuring the antimicrobial activity against the sensitive strain Micrococcus flavus
In previous attempts to characterize the specificity of NisP, the in vivo cleavage was based on the detection of the antimicrobial activity and the detection of mature lantibiotic compared to that of the prelantibiotic
Summary
Lanthipeptides are posttranslationally modified peptides that contain dehydrated amino acids and (methyl)lanthionine residues (Knerr and van der Donk, 2012; Arnison et al, 2013). Lantibiotics are those lanthipeptides that have significant antimicrobial activity, namely lanthipeptide classes I and II. The protease NisP cleaves off the leader peptide releasing mature nisin (Kuipers et al, 1993) In this process, the leader peptide of nisin serves as a recognition motif for the modification enzymes and the transporter and keeps the fully modified prenisin inactive until it is removed (Kuipers et al, 1993; van der Meer et al, 1993; Oman and van der Donk, 2010; Khusainov et al, 2011, 2013; Plat et al, 2011, 2013; Khusainov and Kuipers, 2012; Abts et al, 2013; Yang and van der Donk, 2013)
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