Amino Acids Lanthionine Research Articles

P0095MOLECULAR MECHANISMS OF THE CARDIOVASCULAR EFFECTS OF LANTHIONINE, A NEW UREMIC TOXIN, AND ITS INTERACTIONS WITH THE REDOX MICROENVIRONMENT

Abstract Background and Aims The non-proteinogenic amino acid lanthionine is a byproduct of the biosynthesis of hydrogen sulfide (H2S), an endogenously produced gas with cardiovascular properties). Lanthionine concentration is increased in uremia and it has been proposed as a new uremic toxin. In the zebrafish model, lanthionine induces effects on cardiac embriogenesis, arrhythmia, and locomotor alterations. Some effects are counteracted by glutathione, the well known antioxidant. In a human endothelial cell model, lanthionine significantly reduces H2S release, both protein content and glutathionylation of cystathione beta-synthase (CBS), one of the main H2S-producing enzymes, miR-200c and miR-423 levels, as well as vascular endothelial growth factor expression, while it also increases intracellular calcium levels. We investigated in the present abstract the actions of glutathione on some of lanthionine effects on zebrafish and endothelial cells. We utilized what is considered to be a more stable form of glutathione, acetylglutathione (AcGSH). Method Zebrafish behavior and heart size were analyzed by the Danio Vision system and confocal microscopy, respectively. Gene and protein expression were analyzed usimg qPCR and Western Blot, respectively. CBS glutathionylation has been assessed by immunoprecipitation with a magnetic beads-coated anti-GSH. Interleukins levels were evaluated by ELISA. Lanthionine acetylation was evaluated by LC/MS. Results In zebrafish, we demonstrated that AcGSH significantly increases heart size and the expression of atrium specific proteins. Increased heart rate and heart rhythm plasticity determined by AcGSH were partially counteracted by lanthionine. Monitoring of larval movements showed that this behavior was affected by AcGSH supplementation. In endothelial cells, AcGSH increased H2S release, and, utilized in combination with lanthionine, it was able to partially counteract its effects on H2S release. AcGSH was able to increase CBS glutathionylation and to offset lanthionine effects on glutathionylation. In addition, it increased tubulin, histone, and NFKB acetylation and IL-8 and IL-13 levels. We also demonstrated with mass spectrometry that lanthionine was non-enzymatically acetylated by AcGSH, suggesting a possible mechanism. Conclusion In conclusion, our findings support the notion that lanthionine is a uremic toxin, which is strongly modified in its effects by the redox microenvironment.

Disulphide bonds and cross‐linked amino acids may affect amino acid utilization in feather meal fed to rainbow trout (Oncorhynchus mykiss)

A digestibility trial was carried to assess the apparent digestibility coefficient (ADC) of crude protein (CP) and amino acids (AA) in two feather meals (FeM) of different origin (FeM1 and FeM2) and in their enzymatically pre-treated ingredient stream (PTFeM1 and PTFeM2) in rainbow trout. The ADC of CP was 11%–17% higher (p < 0.05) in the PTFeMs than in their FeM counterparts. There was no difference (p > 0.05) of ADC of CP among FeMs and among PTFeMs. The FT-Raman analysis showed a reduction in concentration and conformational stability of disulphide bonds in the PTFeMs when compared to their FeM counterparts. Moreover, important discrepancies in disulphide concentration were observed between FeMs and between PTFeMs. These observations suggest that the ADC of CP in FeM is not only governed by disulphide bond content and other variables should be considered. Higher concentrations of the cross-linked amino acids (CLAA) lanthionine, lysinoalanine and β-aminoalanine were measured in FeM1 and PTFeM1 as compared to FeM2 and PTFeM2 respectively. Previous studies have suggested inverse relationships between CLAAs and the digestibility as well as bioavailability of AAs in feed ingredients. A previous study conducted in our laboratory showed a lower (p < 0.05) bioavailability of arginine in FeM1 and PTFeM1 than in FeM2 and PTFeM2, respectively, in rainbow trout. Our results suggest that (a) the digestibility of AAs in FeM may be affected by disulphide bond content and the presence of CLAAs, and (b) CLAA content may be indicator of bioavailability of AAs in FeM.

Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale.

The rise of antibiotic resistance demands the acceleration of molecular diversification strategies to inspire new chemical entities for antibiotic medicines. We report here on the large-scale engineering of ribosomally synthesized and post-translationally modified antimicrobial peptides carrying the ring-forming amino acid lanthionine. New-to-nature variants featuring distinct properties were obtained by combinatorial shuffling of peptide modules derived from 12 natural antimicrobial lanthipeptides and processing by a promiscuous post-translational modification machinery. For experimental characterization, we developed the nanoFleming, a miniaturized and parallelized high-throughput inhibition assay. On the basis of a hit set of >100 molecules, we identified variants with improved activity against pathogenic bacteria and shifted activity profiles, and extrapolated design guidelines that will simplify the identification of peptide-based anti-infectives in the future.

Lantibiotic production is a burden for the producing staphylococci

Lantibiotics are antimicrobial peptides that contain non-proteinogenic amino acids lanthionine and 3-methyllanthionine and are produced by Gram-positive bacteria. Here we addressed the pros and cons of lantibiotic production for its producing strains. Two staphylococcal strains, S. gallinarum Tü3928 and S. epidermidis Tü3298 producing gallidermin and epidermin respectively were selected. In each of these parental strains, the structural genes gdmA and epiA were deleted; all the other biosynthetic genes including the immunity genes were left intact. Comparative analysis of the lantibiotic-producing strains with their non-producing mutants revealed that lantibiotic production is a burden for the cells. The production affected growth, caused release of ATP, lipids and increased the excretion of cytoplasmic proteins (ECP). The epidermin and gallidermin immunity genes were insufficient to protect the cells from their own product. Co-cultivation studies showed that the ΔgdmA mutant has an advantage over the parental strain; the latter was outcompeted. On the one hand, the production of staphylococcal lantibiotics is beneficial by suppressing competitors, but on the other hand they impose a burden on the producing-strains when they accumulate in higher amounts. Our observations explain why antibiotic-producing strains occur as a minority on our skin and other ecological niches, but retain corresponding antibiotic resistance.

Zebrafish, a Novel Model System to Study Uremic Toxins: The Case for the Sulfur Amino Acid Lanthionine.

The non-proteinogenic amino acid lanthionine is a byproduct of hydrogen sulfide biosynthesis: the third endogenous vasodilator gas, after nitric oxide and carbon monoxide. While hydrogen sulfide is decreased in uremic patients on hemodialysis, lanthionine is increased and has been proposed as a new uremic toxin, since it is able to impair hydrogen sulfide production in hepatoma cells. To characterize lanthionine as a uremic toxin, we explored its effects during the early development of the zebrafish (Danio rerio), a widely used model to study the organ and tissue alterations induced by xenobiotics. Lanthionine was employed at concentrations reproducing those previously detected in uremia. Light-induced visual motor response was also studied by means of the DanioVision system. Treatment of zebrafish embryos with lanthionine determined acute phenotypical alterations, on heart organogenesis (disproportion in cardiac chambers), increased heart beating, and arrhythmia. Lanthionine also induced locomotor alterations in zebrafish embryos. Some of these effects could be counteracted by glutathione. Lanthionine exerted acute effects on transsulfuration enzymes and the expression of genes involved in inflammation and metabolic regulation, and modified microRNA expression in a way comparable with some alterations detected in uremia. Lanthionine meets the criteria for classification as a uremic toxin. Zebrafish can be successfully used to explore uremic toxin effects.

Nisin and its food application

Bacteriocins are ribosomally synthesized polypeptides which possess antimicrobial activity and are produced by Lactic Acid Bacteria (LAB). Nisin is the most used bacteriocin in food products, is produced by the lactic acid bacterium Lactococcus lactis subsp lactis and its amino-acids are classified to the Lantibiotics group. The peptide chain of this group contains the amino acids lanthionine and a- methyl-lanthionine. Nisin is typically active against Gram-positive bacteria. The mechanism of action of nisin involves mainly the formation of transient pores in the cytoplasmic membrane of the target organism, with loss of membrane potential and leakage of intracellular metabolites resulting in the cell death. The inability of nisin to attack easily the Gram-negative bacteria is due to their protective outer membrane. The demand of consumers for more "natural" additives to food products has increased the interest for the use of nisin in the food industry. Nisin is the only bacteriocin authorized for use as food preservative in over 50 countries. In the EU nisin is approved as food additive in foods like cream and cheese under the E 234 label. A lot of research work has proved the antibacterial activity of nisin against food born pathogens, like Listeria spp., Bacillus spp., Staphylococcus aureus, Clostridium perfrigens, with encouraging results. It is important to note the synergistic action of nisin against food born pathogens with other acting compounds, like chelate factors. Nisin is successfully applied to packaged foods by means of its incorporation or surface absorption to packaging material.

Neuroprotective and neurotrophic effects of Lanthionine Ketimine Ester.

Lanthionine ketimine ethyl ester (LKE) is a synthetic derivative of the naturally occurring amino acid lanthionine ketimine. We previously showed that LKE reduced clinical signs in a mouse model of multiple sclerosis (MS) associated with reductions in axonal damage; however, whether LKE has direct beneficial actions on mammalian neuronal cells was not examined. In the current study, we tested the effects of LKE in SH-SY5Y human neuronal cells and in primary mouse cerebellar granule neurons. In both cell types, LKE dose-dependently reduced the cell death that occurred spontaneously followed a change in media. LKE also reduced cell death due to glutamate excitoxicity, accompanied by a reduction in production of reactive oxygen species. LKE induced neuritogenesis in both undifferentiated SH-SY5Y cells and in primary neuron, increasing process numbers and lengths. These results demonstrate that direct neuroprotective and neurotrophic effects of LKE likely contribute to its beneficial actions in vivo.

Dual peptide system wipes out resistant microbes

Lantibiotics are polypeptide antibiotics that feature one or more thioether-linked ring substructures. They form when bacteria modify a precursor polypeptide by dehydrating serine or threonine residues, followed by cyclizing them with a cysteine thiol. This process forms the amino acid lanthionine or methyllanthionine, which makes up one side of the ring (shown). A newly identified lantibiotic, dubbed bicereucin, is unusual in that it features two separate polypeptides that work in tandem to kill bacterial strains such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, report Liujie Huo and Wilfred A. van der Donk of the University of Illinois, Urbana-Champaign (J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b02513). Working with Bacillus cereus strain SJ1, Huo and van der Donk identified the two bicereucin polypeptides, which they call Bsjα and Bsjβ. Bsjα lacks thioether-connected rings and consequently is not a lantibiotic, whereas Bsjβ is a traditional lantibiotic with one thioether ring. The researchers found

The Supersized Class III Lanthipeptide Stackepeptin Displays Motif Multiplication in the Core Peptide.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides bearing the characteristic amino acids lanthionine and/or labionin. Here, we report on the discovery and characterization of the stackepeptins, produced by the Actinomycete Stackebrandtia nassauensis DSM-44728(T). The stackepeptins are the first supersized class III lanthipeptides to be discovered. Unlike other class III lanthipeptides, they consist of three lanthionine/labionin moieties instead of two. In this study, both in vivo and in vitro maturation of the peptides have been investigated. Studies involving the wild type strain showed culture medium-dependent production of three stackepeptins consisting of one common N-terminal labionin ring and varying dehydration and cyclization patterns in the C-terminal rings. On the other hand, in vitro assessment of the heterologously expressed modifying enzyme StaKC, yielded one major product with an N-terminal lanthionine and C-terminal labionins. The discrepancy between in vivo and in vitro processing was discovered to be sequence-dependent and also implies that in vivo processing is facilitated by additional factors in the cell. Furthermore, a Ser → Ala scan revealed the importance of C-terminal ring formation for full in vitro maturation of the stackepeptins. StaKC showed promiscuity toward the phosphorylating cosubstrate with a significant preference for purine nucleotides. Finally, in contrast to other known class III lanthipeptides, in vitro experiments showed that the leader peptide might not be required for partial dehydration by the modifying enzyme.

Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides.

Lantibiotics are ribosomally synthesized antimicrobial peptides with substantial posttranslational modifications. They are characterized by the unique amino acids lanthionine and methyllanthionine, which are introduced by dehydration of Ser/Thr residues and linkage of the resulting dehydrated amino acids with Cys residues. BLAST searches using the mersacidin biosynthetic enzyme (MrsM) in the NCBI database revealed a new class II lantibiotic gene cluster in Bacillus pseudomycoides DSM 12442. Production of an antimicrobial substance with activity against Gram-positive bacteria was detectable in a cell wash extract of this strain. The substance was partially purified, and mass spectrometric analysis predicted a peptide of 2,786 Da in the active fraction. In order to characterize the putative lantibiotic further, heterologous expression of the predicted biosynthetic genes was performed in Escherichia coli. Coexpression of the prepeptide (PseA) along with the corresponding modification enzyme (PseM) resulted in the production of a modified peptide with the corresponding mass, carrying four out of eight possible dehydrations and supporting the presence of four thioether and one disulfide bridge. After the proteolytic removal of the leader, the core peptide exhibited antimicrobial activity. In conclusion, pseudomycoicidin is a novel lantibiotic with antimicrobial activity that was heterologously produced in E. coli.

Structure and biosynthesis of carnolysin, a homologue of enterococcal cytolysin with D-amino acids.

Lantibiotics are a group of highly post-translationally modified bacterial antimicrobial peptides characterized by the presence of the thioether-containing amino acids lanthionine and methyllanthionine. Carnobacterium maltaromaticum C2 was found to produce a two-component lantibiotic homologous to enterococcal cytolysin. Through tandem mass spectrometry and NMR spectroscopy, the post-translational modifications of carnolysin were established, and the topologies of the lanthionine and methyllanthionine rings were determined. Chiral GC-MS analysis revealed that, like cytolysin, carnolysin contained lanthionine and methyllanthionine residues of unusual stereochemistry. Carnolysin, unlike cytolysin, was shown to contain d-alanine and unprecedented D-aminobutyrate derived from serine and threonine, respectively. Carnolysin was heterologously expressed in Escherichia coli, demonstrating that reductase CrnJ is involved in the formation of the D-amino acids.

Characterization of Amylolysin, a Novel Lantibiotic from Bacillus amyloliquefaciens GA1

BackgroundLantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity.Methodology/Findings Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact in vitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus.Conclusion/SignificanceApart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens.

The Lantibiotic Peptide Labyrinthopeptin A1 Demonstrates Broad Anti-HIV and Anti-HSV Activity with Potential for Microbicidal Applications

Lantibiotics are peptides, produced by bacteria, that contain the noncanonical amino acid lanthionine and many of them exhibit antibacterial activities. The labyrinthopeptin A1 (LabyA1) is a prototype peptide of a novel class of carbacyclic lantibiotics. Here, we extensively evaluated its broad-spectrum activity against HIV and HSV in vitro, studied its mechanism of action and evaluated potential microbicidal applications. LabyA1 exhibited a consistent and broad anti-HIV activity (EC50s: 0.70–3.3 µM) and anti-HSV activity (EC50s: 0.29–2.8 µM) in cell cultures. LabyA1 also inhibited viral cell-cell transmission between persistently HIV-infected T cells and uninfected CD4+ T cells (EC50∶2.5 µM) and inhibited the transmission of HIV captured by DC-SIGN+-cells to uninfected CD4+ T cells (EC50∶4.1 µM). Time-of-drug addition studies revealed that LabyA1 acts as an entry inhibitor against HIV and HSV. Cellular and virus binding studies combined with SPR/FLIPR technology showed that LabyA1 interacted with the HIV envelope protein gp120, but not with the HIV cellular receptors. LabyA1 also demonstrated additive to synergistic effects in its anti-HIV-1 and anti-HSV-2 activity with anti(retro)viral drugs in dual combinations such as tenofovir, acyclovir, saquinavir, raltegravir and enfuvirtide. LabyA1 can be considered as a novel lead peptide as it had profound antiviral activity against HIV and HSV. Pre-treatment of PBMCs with LabyA1 neither increased the expression of the activation markers CD69 and CD25, nor enhanced HIV replication, nor significantly induced various inflammatory cytokines/chemokines. LabyA1 also did not affect the growth of vaginal Lactobacilli populations. Based on the lack of toxicity on the vaginal Lactobacillus strains and its synergistic/additive profile in combination with clinically approved anti(retro)virals, it deserves further attention as a potential microbicide candidate in the prevention of sexual transmitted diseases.

Chemical Synthesis of the Lantibiotic Lacticin 481 Reveals the Importance of Lanthionine Stereochemistry

Lantibiotics are a family of antibacterial peptide natural products characterized by the post-translational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single naturally occurring stereochemical configuration for each of these cross-links was known. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing cross-links with non-native stereochemical configurations. Biological evaluation revealed that these alterations abolished the antibacterial activity in all of the analogues, revealing the critical importance of the enzymatically installed stereochemistry for the biological activity of lacticin 481.

Characterization of New Class III Lantibiotics—Erythreapeptin, Avermipeptin and Griseopeptin from Saccharopolyspora erythraea, Streptomyces avermitilis and Streptomyces griseus Demonstrates Stepwise N‐Terminal Leader Processing

Lantibiotics are a large group of ribosomally synthesized peptides post-translationally modified to incorporate the amino acid lanthionine. They are classified, according to their biosynthetic pathway and bioactivity, into three major subtypes. Of Actinomycetes type III lantibiotics, only four peptides (SapB, SapT, LabA1, and LabA2) have been described and structurally characterized, although homologous gene clusters are abundant in other Actinomycetes. All these gene clusters share a similar architecture with a characteristic Ser/Ser/Cys motif in precursor peptides, which has previously been suggested to act as a precursor for lanthionine (SapB) and labionin (LabA2) rings. Mass spectrometry screening led to the discovery and characterization of three new representatives of type III lantibiotics: Avermipeptin (Avi), Erythreapeptin (Ery), and Griseopeptin (Gri) from Streptomyces avermitilis DSM 46492, Saccharopolyspora erythraea NRRL 2338, and Streptomyces griseus DSM 40236, respectively. Apart from the assignment of these peptides to their corresponding gene clusters, additional investigations on Avi, Ery and Gri peptides indicate stepwise leader processing by putative aminopeptidase-like protease(s), thus yielding mixtures of differently N-terminal-processed lantibiotic peptides. Similar peptide processing was observed for a heterologously expressed eryth biosynthetic gene cluster expressed in a Streptomyces host system. Remarkably, all isolates of the new type III lantibiotics contain both the amino acids lanthionine and labionin, thus implying dual-mode cyclase activity of the processing lyase-kinase-cyclase enzymes. These findings have implications for the structures and maturation of other type III lantibiotics from Actinomycetes.

Interaction of Type A Lantibiotics with Undecaprenol-Bound Cell Envelope Precursors

Lantibiotics are a unique group within the antimicrobial peptides characterized by the presence of thioether amino acids (lanthionine and methyllanthionine). These peptides are produced by and primarily act on Gram-positive bacteria exerting multiple activities at the cytoplasmic membrane of susceptible strains. Previously, the cell wall precursor lipid II was identified as the molecular target for the prototype lantibiotic nisin. Binding and sequestration of lipid II blocks the incorporation of the central cell wall precursor into the growing peptidoglycan network, thereby inhibiting the formation of a functional cell wall. Additionally, nisin combines this activity with a unique target-mediated pore formation, using lipid II as a docking molecule. The interaction with the pyrophosphate moiety of lipid II is crucial for nisin binding. We show that, besides binding to lipid II, nisin interacts with the lipid intermediates lipid III (undecaprenol-pyrophosphate-N-acetyl-glucosamine) and lipid IV (undecaprenol-pyrophosphate-N-acetyl-glucosamine-N-acetyl-mannosamine) of the wall teichoic acid (WTA) biosynthesis pathway. Binding of nisin to the precursors was observed at a stoichiometry of 2:1. The specific interaction with WTA precursors further promoted target-mediated pore formation in artificial lipid bilayers. Specific interactions with lipid III and lipid IV could also be demonstrated for related type A lantibiotics, for example, gallidermin, containing the conserved lipid-II-binding motif.

The Synthesis of Active and Stable Diaminopimelate Analogues of the Lantibiotic Peptide Lactocin S

Lantibiotic peptides are potent antimicrobial compounds produced by Gram-positive bacteria. They can be used in food preservation, and some also show potential for clinical applications. Unfortunately, some of these peptides can be susceptible to inactivation by oxidation of the sulfur-containing amino acid lanthionine, limiting their use. Here we describe the synthesis and testing of diaminopimelate analogues of the lantibiotic lactocin S. These analogues were designed to improve the oxidative stability of the peptide by replacing the sulfur in lanthionine with a methylene unit. Lanthionine was systematically replaced with diaminopimelate during solid-phase peptide synthesis to produce several analogues. One analogue, A-DAP lactocin S, was found to retain full biological activity in addition to displaying increased stability. This is the first time a synthetic lanthionine ring analogue of a lantibiotic has retained natural activity levels. This methodology is potentially very promising for use in producing more stable, medically relevant lantibiotics.

Leader Peptide-Directed Processing of Labyrinthopeptin A2 Precursor Peptide by the Modifying Enzyme LabKC

Lantibiotics are peptide antibiotics, realizing their unique secondary structure by posttranslational modifications, the most important one being the formation of the characteristic amino acid lanthionine. Like other ribosomal peptide antibiotics, they are synthesized with an N-terminal leader peptide important for posttranslational processing by modifying enzymes; after peptide maturation, the leader peptide is proteolytically cleaved off. Numerous studies of the leader peptides of class I and II lantibiotics already showed their crucial role in recognition, self-immunity, and extracellular transport. The recently described labyrinthopeptins, members of the family of class III lantibiotics, exhibit the characteristic novel amino acid labionin, which was revealed by elucidation of the structure of labyrinthopeptin A2. The assembly of the labionin motif in the linear peptide chain is mediated by the lyase-kinase-cyclase-type enzyme LabKC through a serine side chain phosphorylation with GTP, elimination of the phosphate group, and a subsequent 2-fold Michael-type addition cyclization. In this work, we systematically investigated for the first time the importance of the leader peptide in the processing of class III lantibiotics using the example of the labyrinthopeptin A2 precursor peptide. In vitro studies with synthetic leader peptide analogues revealed that a conserved N-terminal hydrophobic patch on a putative helical structure is required for the proper peptide processing by the modifying enzyme LabKC. On the other hand, studies showed that the C-terminal part of the leader peptide serves as a spacer between the binding site and active sites for phosphorylation and elimination, thus restricting the number of hydroxy amino acid side chains that could undergo dehydration. Finally, a model for the peptide recognition and processing by the LabKC has been postulated.

Substrate Recognition and Specificity of the NisB Protein, the Lantibiotic Dehydratase Involved in Nisin Biosynthesis

Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl)lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Light scattering analysis demonstrated that purified NisB is a dimer in solution. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified prenisin binds to NisB with an affinity of 1.05 ± 0.25 μm, whereas the dehydrated and the fully modified derivatives bind with respective affinities of 0.31 ± 0.07 and 10.5 ± 1.7 μm. The much lower affinity for the fully modified prenisin was related to a >20-fold higher off-rate. For all three peptides the stoichiometry of binding was 1:1. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.

Nine Post-translational Modifications during the Biosynthesis of Cinnamycin

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides that are characterized by the thioether cross-linked amino acids lanthionine (Lan) and methyllanthionine (MeLan). Cinnamycin is a 19 amino acid lantibiotic that contains one Lan and two MeLan. Cinnamycin also contains an unusual lysinoalanine (Lal) bridge formed from the ε-amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-l-aspartic acid resulting from the hydroxylation of l-aspartate at position 15. These modifications are critical in mediating the interactions of cinnamycin with its target, phosphatidylethanolamine. Recently, the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005 was reported. Herein, we investigated the biosynthetic machinery using both in vitro studies and heterologous expression in Escherichia coli. CinX is an α-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide CinA. In addition, CinM catalyzes dehydration of four Ser and Thr residues and subsequent cyclization of Cys residues to form the three (Me)Lan bridges. The order of the post-translational modifications catalyzed by CinM and CinX is interchangeable in vitro. CinX did not require the leader sequence at the N-terminus of CinA for activity, but the leader peptide was necessary for CinM function. Although CinM dehydrated serine 6, it did not catalyze the formation of Lal. A small protein encoded by cinorf7 is critical for the formation of the cross-link between Lys19 and dehydroalanine 6 as shown by coexpression studies of CinA, CinM, CinX, and Cinorf7 in E. coli.