Correction: The Presence of Modifiable Residues in the Core Peptide Part of Precursor Nisin Is Not Crucial for Precursor Nisin Interactions with NisB- and NisC
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PROVIDER: S-EPMC3997513 | biostudies-literature | 2014 Jan
REPOSITORIES: biostudies-literature
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Project description:Nisin is a posttranslationally-modified antimicrobial peptide that has the ability to induce its own biosynthesis. Serines and threonines in the modifiable core peptide part of precursor nisin are dehydrated to dehydroalanines and dehydrobutyrines by the dehydratase NisB, and subsequently cysteines are coupled to the dehydroamino acids by the cyclase NisC. In this study, we applied extensive site-directed mutagenesis, together with direct binding studies, to investigate the molecular mechanism of the dehydratase NisB. We use a natural nisin-producing strain as a host to probe mutant-NisB functionality. Importantly, we are able to differentiate between intracellular and secreted fully dehydrated precursor nisin, enabling investigation of the NisB properties needed for the release of dehydrated precursor nisin to its devoted secretion system NisT. We report that single amino acid substitutions of conserved residues, i.e., R83A, R83M, and R87A result in incomplete dehydration of precursor nisin and prevention of secretion. Single point NisB mutants Y80F and H961A, result in a complete lack of dehydration of precursor nisin, but do not abrogate precursor nisin binding. The data indicate that residues Y80 and H961 are directly involved in catalysis, fitting well with their position in the recently published 3D-structure of NisB. We confirm, by in vivo studies, results that were previously obtained from in vitro experiments and NisB structure elucidation and show that previous findings translate well to effects seen in the original production host.
| S-EPMC4341554 | biostudies-literature
Project description:The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg(2+), and the ribosomal/membrane fraction of bacterial cell extract. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A, that result in multiple glutamylations of the NisA substrate. Glutamylation was observed during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results in dehydrated NisA, suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin.
| S-EPMC3645518 | biostudies-literature
Project description:The dehydratase NisB performs stepwise tRNAGlu-dependent glutamylation of Ser/Thr residues and subsequent glutamate elimination to effect eight dehydrations in the biosynthesis of the antibacterial peptide nisin. Its substrate, NisA, bears a C-terminal core peptide that is modified and an N-terminal leader peptide (LP) that is not modified but that is required for efficient dehydration. To elucidate the mechanism of LP-NisB interactions during dehydration, we engineered a disulfide that covalently links the NisA LP to NisB. The enzyme fully dehydrated tethered NisA, confirming the functional LP binding site and supporting a mechanism where NisB uses a single LP binding site for glutamylation and elimination. We also show an order of NisA and tRNAGlu binding to NisB that enables dehydration.
| S-EPMC5901694 | biostudies-literature
Project description:Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serine and threonine residues, yielding dehydroalanine and dehydrobutyrine, respectively. Cyclase-catalyzed coupling of the formed dehydroresidues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow discovery of target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations on installing lanthionines in non-natural substrates. The goal of the present study was to obtain a lanthipeptide dehydratase with the capacity to dehydrate substrates that are unsuitable for the nisin dehydratase NisB. We report high-throughput screening for tailored specificity of intracellular, genetically encoded NisB dehydratases. The principle is based on the screening of bacterially displayed lanthionine-constrained streptavidin ligands, which have a much higher affinity for streptavidin than linear ligands. The designed NisC-cyclizable high-affinity ligands can be formed via mutant NisB-catalyzed dehydration but less effectively via wild-type NisB activity. In Lactococcus lactis, a cell surface display precursor was designed comprising DSHPQFC. The Asp residue preceding the serine in this sequence disfavors its dehydration by wild-type NisB. The cell surface display vector was coexpressed with a mutant NisB library and NisTC. Subsequently, mutant NisB-containing bacteria that display cyclized strep ligands on the cell surface were selected via panning rounds with streptavidin-coupled magnetic beads. In this way, a NisB variant with a tailored capacity of dehydration was obtained, which was further evaluated with respect to its capacity to dehydrate nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity.
| S-EPMC7309312 | biostudies-literature
Project description:Nisin is the most prominent and applied bacteriocin that serves as a model for class I lantibiotics. The nisin leader peptide importantly determines interactions between precursor nisin and its modification enzymes NisB and NisC that mature nisin posttranslationally. NisB dehydrates serines and threonines, while NisC catalyzes the subsequent coupling of the formed dehydroamino acids to form lanthionines. Currently, little is known about how the nisin leader interacts with NisB and even less is known about its interactions with NisC. To investigate the nisin leader peptide requirements for functional interaction with the modification enzymes NisB and NisC, we systematically replaced six regions, of 2-4 amino acids each, with all-alanine regions. By performing NisB and NisC co-purification studies with these mutant leader peptides, we demonstrate that the nisin leader regions STKD(-22-19), FNLD(-18-15) and PR(-2-1) importantly contribute to the interactions of precursor nisin with both NisB and NisC, whereas the nisin leader region LVSV(-14-11) additionally contributes to the interaction of precursor nisin with NisC.
| S-EPMC3678300 | biostudies-literature
Project description:Nisin produced by Lactococcus lactis 6F3 is used as a food preservative and is the most important member of a group of peptide-antibiotics containing lanthionine bridges (lantibiotics) (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). Nisin is ribosomally synthesized, and its structural gene, nisA, encodes a prepeptide that is posttranslationally modified, revealing the active lantibiotic (C. Kaletta and K.-D. Entian, J. Bacteriol. 171:1597-1601, 1989). Adjacent to nisA, the additional genes nisB, nisT, and nisC were identified. Over their entire sequences, these genes were homologous to genes recently identified as important for the biosynthesis of lantibiotics, that is, subtilin from Bacillus subtilis ATCC 6633 and epidermin from Staphylococcus epidermidis Tü 3298. Genes nisB, nisT, and nisC corresponded to open reading frames of 993, 600, and 418 amino acid residues, respectively. The nisT open reading frame is homologous to proteins of the HlyB (hemolysin B protein of Escherichia coli) subfamily. Proteins of this subfamily are responsible for the secretion of a variety of compounds, including large polypeptides, polysaccharides, and anti-drug tumors, indicating that NisT may be involved in nisin transport. Northern (RNA) blot analysis revealed a 0.3-kb transcript for the nisA structural gene, and the transcriptional start point of the nisA gene was determined by primer extension. Additionally, a mRNA of at least 3 kb was identified by using a hybridization probe specific to nisB. Antibodies were raised against the NisB protein, and Western blot (immunoblot) analysis revealed a molecular weight of about 115 kDa, which is in accordance with the theoretical protein size of 117.5 kDa as calculated from the nisB open reading frame. Several amphipathic transmembrane alpha-helices indicated that NisB is associated with the membrane. This was confirmed by preparing L. lactis vesicles. The NisB protein was tightly associated with the vesicle fraction and was released by sodium dodecyl sulfate treatment only. These results suggest that NisB is membrane associated and that nisin biosynthesis occurs at the cell membrane.
| S-EPMC183167 | biostudies-other
Project description:The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.
| S-EPMC1280264 | biostudies-literature
Project description:An alanine scan of Lys10-teixobactin reveals that a cationic residue at position 10 is not necessary for antibiotic activity and that position 3 tolerates substitution without loss of activity. An unexpected correlation between poor aqueous solubility and better antibiotic activity of the teixobactin analogues is observed.
| S-EPMC6361391 | biostudies-literature
Project description:Histidine kinase (HK) NisK is well known to sense lantibiotic nisin for regulating the biosynthesis of nisin. NisK possesses two trans-membrane segments and a large extracellular region and nisin contains 34 amino acids with five lanthionine rings. Unlike most peptide sensing HK with multi trans-membrane segments, NisK is a representative of a group of rarely reported HK that sense peptide as ligand. To reveal how NisK senses nisin molecule to regulate nisin biosynthesis, we constructed a reporter Lactococcus lactis strain with nisRK constitutively expressed and a reporter gene lacZ expressed under the control of promoter P nisA . We showed that the extracellular region of NisK was involved in recognizing nisin. Conserved residues in this group of HK were found in the extracellular region of NisK and mutagenesis of these residues in the reporter strain revealed that several hydrophobic residues including two aromatic residues are crucial for NisK sensing nisin and regulating nisin biosynthesis. Substitutions of hydrophobic regions in NisK extracellular domain showed that the first strand that was rich of hydrophobic amino acids was involved in regulating nisin biosynthesis. A negatively charged residue in the first βstrand also contributed to nisin biosynthesis. Protein binding analyses demonstrated that nisin could not interact with key NisK mutants, indicating these site in the extracellular region of NisK was involved in recognizing nisin.
| S-EPMC5266694 | biostudies-literature
Project description:This work evaluates the hypothesis that proteins with an identical supersecondary structure (SSS) share a unique set of residues--SSS-determining residues--even though they may belong to different protein families and have very low sequence similarities. This hypothesis was tested on two groups of sandwich-like proteins (SPs). Proteins in each group have an identical SSS, but their sequence similarity is below the "twilight zone." To find the SSS-determining residues specific to each group, a unique structure-based algorithm of multiple sequences alignment was developed. The units of alignment are individual strands and loops rather than whole sequences. The algorithm is based on the alignment of residues that form hydrogen bonds between corresponding strands. Structure-based alignment revealed that 30-35% of the positions in the sequences in each group of proteins are "conserved positions" occupied either by hydrophobic-only or hydrophilic-only residues. Moreover, each group of SPs is characterized by a unique set of SSS-determining residues found at the conserved positions. The set of SSS-determining residues has very high sensitivity and specificity for identifying proteins with a corresponding SSS: It is an "amino acid tag" that brands a sequence as having a particular SSS. Thus, the sets of SSS-determining residues can be used to classify proteins and to predict the SSS of a query amino acid sequence.
| S-EPMC2776455 | biostudies-literature