Incorporation of tryptophan analogues into the lantibiotic nisin

The alarming rise of antimicrobial resistance requires antibiotics with unexploited mechanisms. Ideal templates could be antibiotics that target the peptidoglycan precursor lipid II, known as the bacterial Achilles heel, at an irreplaceable pyrophosphate group. Such antibiotics would kill multidrug-resistant pathogens at nanomolecular concentrations without causing antimicrobial resistance. However, due to the challenge of studying small membrane-embedded drug–receptor complexes in native conditions, the structural correlates of the pharmaceutically relevant binding modes are unknown. Here, using advanced highly sensitive solid-state NMR setups, we present a high-resolution approach to study lipid II-binding antibiotics directly in cell membranes. On the example of nisin, the preeminent lantibiotic, we show that the native antibiotic-binding mode strongly differs from previously published structures, and we demonstrate that functional hotspots correspond to plastic drug domains that are critical for the cellular adaptability of nisin. Thereby, our approach provides a foundation for an improved understanding of powerful antibiotics.

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“…3 ). This assumption is supported by mutational studies that showed a twofold activity reduction upon replacing I1 by a tryptophan 55 .…”

Section: Discussionmentioning

confidence: 83%

High-resolution NMR studies of antibiotics in cellular membranes

et al. 2018

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“…The promiscuity of NisBTC and the development of an efficient production system for the modification and export of modified peptides enabled the production of putative lantibiotics from diverse bacteria in L. lactis (Majchrzykiewicz et al, 2010 ; van Heel et al, 2016 ). Moreover, this production system can be extended with additional enzyme modules (i.e., additional modification enzymes found in lantibiotic gene clusters) (van Heel et al, 2013 ) or with non-canonical amino acids (Zhou et al, 2016 ; Baumann et al, 2017 ; Zambaldo et al, 2017 ) that increase the repertoire of unusual amino acids that can be incorporated in vivo in peptides. These findings highlight the large potential of using Synthetic Biology principles in the production of novel lanthipeptides (for a review see Montalbán-López et al, 2012 , 2017 and references therein).…”

Section: Introductionmentioning

confidence: 99%

Specificity and Application of the Lantibiotic Protease NisP

et al. 2018

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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.

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“…18-19 Kuipers also reported the incorporation of tryptophan analogues into Nisin using an auxotrophic strain. 20 With the goal of creating Nisin variants through biomimetic cyclization reactions (Figure 2), we built upon the former system 18 and began by recombinantly expressing wild type (WT) NisA in bacterial strains in which we have previously genetically encoded a large number of ncAAs. The nisA structural gene together with the dehydratase ( nisB ), were inserted into a pRSF-1 vector to afford pRSF-NisA.…”

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confidence: 99%

Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

et al. 2017

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Nisin is a complex lanthipeptide that has broad spectrum antibacterial activity. In efforts to broaden the structural diversity of this ribosomally synthesized lantibiotic, we now report the recombinant expression of Nisin variants that incorporate non-canonical amino acids (ncAAs) at discrete positions. This is achieved by expressing the nisA structural gene, cyclase (nisC) and dehydratase (nisB), together with an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase pair in E.coli. A number of ncAAs with novel chemical reactivity were genetically incorporated into NisA, including an α-chloroacetamide-containing ncAA which allowed for the expression of Nisin variants with novel macrocyclic topologies. This methodology should allow for the exploration of lanthipeptide variants with new or enhanced activities.

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Incorporation of tryptophan analogues into the lantibiotic nisin

Cited by 37 publications

References 37 publications

High-resolution NMR studies of antibiotics in cellular membranes

High-resolution NMR studies of antibiotics in cellular membranes

Specificity and Application of the Lantibiotic Protease NisP

Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

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