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Here we describe bibacillin 1 - a two-component lantibiotic from Bacillus thuringiensis . The peptides that comprise bibacillin 1 are modified by a class II lanthipeptide synthetase Bib1M producing two peptides with non-overlapping ring patterns that are reminiscent of cerecidin and the short component of the enterococcal cytolysin (CylL S "), a virulence factor associated with human disease. Stereochemical analysis demonstrated that each component contains LL-methyllanthionine and DL-lanthionine. The mature bibacillin 1 peptides showed cooperative bactericidal activity against Gram-positive bacteria, including members of ESKAPE pathogens, and weak hemolytic activity. Optimal ratio studies suggest that bibacillin 1 works best when the components are present in a 1:1 ratio, but near optimal activity was observed at ratios strongly favouring one component over the other, suggesting that the two peptides may have different but complementary targets. Mechanism of action studies suggest a lipid II-independent killing action distinguishing bibacillin 1 from two other two-component lantibiotics haloduracin and lacticin 3147. One of the two components of bibacillin 1 showed cross reactivity with the cytolysin regulatory system. These result support the involvement of bibacillin 1 in quorum sensing and raise questions about the impact of CylL S "-like natural products on lanthipeptide expression in diverse bacterial communities., Competing Interests: Conflicts of Interest There are no conflicts to declare.

Bacteriocins are a large family of bacterial peptides that have antimicrobial activity and potential applications as clinical antibiotics or food preservatives. Circular bacteriocins are a unique class of these biomolecules distinguished by a seamless circular topology, and are widely assumed to be ultra-stable based on this constraining structural feature. However, without quantitative studies of their susceptibility to defined thermal, chemical, and enzymatic conditions, their stability characteristics remain poorly understood, limiting their translational development. Here, we produced the circular bacteriocin enterocin NKR-5-3B (Ent53B) in mg/L quantities using a heterologous Lactococcus expression system, and characterized its thermal stability by NMR, chemical stability by circular dichroism and analytical HPLC, and enzymatic stability by analytical HPLC. We demonstrate that Ent53B is ultra-stable, resistant to temperatures approaching boiling, acidic (pH 2.6) and alkaline (pH 9.0) conditions, the chaotropic agent 6 M urea, and following incubation with a range of proteases (i.e., trypsin, chymotrypsin, pepsin, and papain), conditions under which most peptides and proteins degrade. Ent53B is stable across a broader range of pH conditions and proteases than nisin, the most widely used bacteriocin in food manufacturing. Antimicrobial assays showed that differences in stability correlated with differences in bactericidal activity. Overall, this study provides quantitative support for circular bacteriocins being an ultra-stable class of peptide molecules, suggesting easier handling and distribution options available to them in practical applications as antimicrobial agents., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lanthipeptides are intriguing peptides known since 1928, the year of penicillin's discovery. At that time, they were known as lantibiotics due to their (methyl)lanthionine amino acids and antibacterial activity. Their body of knowledge expanded tremendously over the last few years. Our analysis reveals that Bacteroidetes has a high state of clusters encoding the biosynthesis of class I lanthipeptides. We show that some strains of Pedobacter have a number of LanBs/genome comparable to that of some Actinobacteria. The case study selected was Pedobacter lusitanus NL19. Its clusters identified encode LanBs associated with LanCs as well as orphan LanBs. The first are concomitant with LanT transporters typical of class II lanthipeptides (and not class I), making their clusters into a hybrid class I and class II type. So far, this kind of operon was described only once and is involved in the production of pinensins, the first lanthipeptide with antifungal activity. A particular feature of pinensins is their splitted LanBs and we found that these enzymes are also widely encoded in Bacteroides. The function of a high percentage of proteins predicted to play a role in the production of Pedobacter lanthipeptides is unknown. Other major fraction of these proteins is expected to be enrolled in signal-transduction pathways. We demonstrate that the occurrence of lanthipeptides clusters in the genomes of Gram-negative bacteria is higher than previously reported. More importantly, we show that their genetic background is highly diverse, which is an undeniable foreshadowing of novel peptide structures, biochemistry and biological function., Competing Interests: Declaration of Competing Interest The author(s) declare that there are no conflicts of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Plant alpha dioxygenases (PADOX) convert fatty acids to 2-hydroperoxy products that are important in plant signaling pathways. The PADOX amino-terminal domain is distinct from that in other myeloperoxidase-family hemoproteins, and the positional specificity and prosthetic group of PADOX distinguish them from the non-heme iron plant lipoxygenases. The constraints of the PADOX active site on potential substrates are poorly understood and only limited structure-function and mechanistic information is available for these enzymes. We developed several bacterial and insect cell systems for expression of recombinant Arabidopsis thaliana PADOX1 and evaluated the enzyme's substrate and inhibitor profiles and explored the functional role of the amino-terminal domain. Substrate specificity studies gave the following relative oxygenase activity values: linolenate, 1.00; linoleate, 0.95; oleate, 0.84; palmitoleate, 0.69; myristate, 0.23; palmitate, 0.17; and gamma-linolenate, 0.16. Methyl esters of myristate, linoleate and linolenate were not oxygenated. 3-Thiamyristate was the only oxygenase substrate that produced pronounced enzyme self-inactivation during catalysis. 3,4-Dehydromyristate inactivated the oxygenase without appreciable oxygen consumption. Several compounds inhibited oxygenase activity, including catechol (K(i) approximately 90 microM), divalent zinc ion (K(i) approximately 50 microM), N,N,N',N'-tetramethyl-p-phenylenediamine (K(i) approximately 20 microM) and cyanide ion (K(i) approximately 5 microM). Zinc ion did not change the K(m) values for linoleate or oxygen, or the K(i) value for cyanide, indicating that zinc acts at a distinct site from the other compounds. Gel-filtration chromatography revealed considerable variation in oligomeric state of recombinant PADOX1 produced in the various expression systems, but oligomeric state was not correlated with activity. Deletion of the first eight or fourteen PADOX1 residues in a NuSA-PADOX1 fusion protein led to 13 and 83% decreases in activity, respectively, indicating the N-terminal region is important for normal catalytic activity.

[reaction in text] A new method for the synthesis of selenocysteine derivatives and selenocysteine-containing peptides is described. Fmoc-Se-p-methoxybenzylselenocysteine (1) was prepared and used for solid-phase synthesis of peptides with an N-terminal unprotected selenocysteine. Subsequent native chemical ligation with a peptide thioester provided a 17-mer that corresponds to the C-terminus of ribonucleotide reductase with selenocysteine in place of cysteine.

[reaction: see text]. Protein and peptide conjugates such as glycopeptides, prenylated peptides, and lipopeptides play essential roles in biology. A rapid and convergent entry into a variety of these compounds is described. The methodology involves the introduction of a dehydroalanine into peptides and subsequent chemoselective conjugate addition of an appropriate thiolate nucleophile, including farnesylthiolate or thioglycosides.

[reaction: see text] Peptides containing (E)- and (Z)-3-fluorodehydroalanine have been prepared from serine via a fluoro-Pummerer rearrangement. The resulting electrophilic moieties may be useful affinity labels for the identification of the targets of dehydroamino acid containing natural products that act by covalent mechanisms.

Useful methodology is described for the synthesis of dehydroalanine residues (II) within peptides. The unnatural amino acid (Se)-phenylselenocysteine (I) can be incorporated into growing peptide chains via standard peptide synthesis procedures. Subsequent oxidative elimination affords a dehydroalanine at the desired position. The oxidation conditions are mild and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. To illustrate its utility, cyclic lanthionines have been synthesized by this method.

Recent crystallographic and biochemical studies have revealed the existence of numerous novel post-translational modifications within enzyme active sites. These modifications create structural and functional diversity. Although the function and biosynthesis of some of these modifications are well understood, others need further investigation.

Ribonucleotide reductases (RNRs) play a central role in replication and repair by catalyzing the conversion of nucleotides to deoxynucleotides. Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was recently approved by the FDA for treatment of pancreatic cancer, is a potent mechanism-based inhibitor of class I and II RNRs. Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorides and one cytosine. This RNR is composed of two homodimeric subunits: R1 and R2. R1 is the site of nucleotide reduction, and R2 contains the essential diferric-tyrosyl radical cofactor. The mechanism of inactivation depends on the availability of reductant. In the presence of reductant [thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiothreitol], inhibition results from R1 inactivation. In the absence of reductant with prereduced R1 and R2, inhibition results from loss of the essential tyrosyl radical in R2. The same result is obtained with C754S/C759S-R1 in the presence of TR/TRR/NADPH. In both cases, tyrosyl radical loss is accompanied by formation of a new stable radical (0.15-0.25 equiv/RNR). EPR studies in 2H2O, with [U-2H]R1, and examination of the microwave power saturation of the observed signal, indicate by process of elimination that this new radical is nucleotide-based. In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a new protein-associated chromophore under any conditions. The requirement for reductant in the R1 inactivation pathway, the lack of chromophore on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivation not previously observed with other 2'-substituted nucleotide inhibitors of RNR. This unique mode of inactivation is proposed to be responsible for its observed clinical efficacy.

Incubation of the C225S mutant of the R1 subunit of ribonucleotide reductase from Escherichia coli with the R2 subunit and nucleoside diphosphates leads to fragmentation of the polypeptide backbone of R1 [Mao, S. S., Holler, T. P., Bollinger, J.M., Jr., Yu, G. X., Johnston, M.I., & Stubbe, J. (1992) Biochemistry 31, 9744--9751]. The 26 and 60 kDa cleavage fragments were purified to homogeneity. The 26 kDa polypeptide was digested with Lys-C, and the peptides were partially purified by RP-HPLC. Mass spectrometric analysis (MALDI-TOF) of the HPLC fractions allowed the identification of the C-terminal peptide. The molecular mass of this peptide (2176) revealed that serine-224 constitutes its C-terminus, and further analysis of the distribution of its monoisotopic masses by FAB-MS indicated that Ser224 possesses a carboxamide rather than a carboxylate group. Treatment of the 60 kDa cleavage fragment with cyanogen bromide and subsequent MALDI-TOF analysis of the partially RP-HPLC purified peptides yielded a fraction containing its N-terminal peptide. This peptide was digested with trypsin, and the digestion mixture was purified by HPLC. Analysis of the fractions by MALDI-TOF identified the N-terminal peptide and determined a mass of 2222. This mass suggested valine 226 was the N-terminal residue (modified by an adduct of 28 mass units). Larger amounts of the C-terminal tetrapeptide of the 60 kDa fragment (V226LIE229) were obtained by complete digestion of the crude reaction mixture with endoproteinase Glu-C. The peptide mixture was then purified on an immunoadsorbent column containing immobilized antibodies raised against a synthetic peptide with the sequence KVLIE. After elution of the affinity-bound peptide, it was analyzed by CID-MS verifying that an adduct of 28 mass units was attached to valine 226. These results indicated that the amino group of Val226 is formylated. The localization of the residues at the cleavage site of C225SR1 provides a biochemical identification of the active site region of the R1 subunit of RDPR from E.coli. The details of the mechanism of cleavage remain to be elucidated.

Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. (E)- and (Z)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate (FMCDP) are time dependent inactivators of this protein, with approximately 1.5 equiv being sufficient for complete loss of catalytic activity. Inactivation results from loss of the essential tyrosyl radical on R2 and alkylation of R1. Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accompanied by formation of a new, substrate-based radical. Experiments using [6'-14C]-(E)-FMCDP and [5-3H]-(E)-FMCDP reveal that alkylation of R1 is accompanied by release of 0.5 equiv of cytosine and 1.4 equiv of fluoride ion. When R1 is denatured subsequent to inactivation, approximately 1 equiv of label per R1 is observed only in studies carried out with [14C]FMCDP. Under these same conditions with [3H]FMCDP, 1.5 equiv of radiolabel is detected as cytosine. Inactivation of R1 thus results from alkylation by the sugar moiety of FMCDP. While studies to isolate the alkylated amino acid on R1 were unsuccessful, studies using a variety of site-directed mutants of R1 (C462S, C225S, C754/759S, C439S, and E441Q) indicate that E441 or possibly C439 is the modified residue. Inactivation is accompanied by rapid formation of a new chromophore with a lambda max at 334 nm. Dithiothreitol does not protect the enzyme against inactivation by FMCDP, although it does prevent chromophore formation. Two possible mechanisms are proposed to accommodate these experimental observations.

Ribonucleotide reductases catalyze a key step in DNA biosynthesis, using a diverse array of unprecedented metallo-cofactors to generate a transient protein radical that initiates nucleotide reduction. The new understanding of the chemistry and biochemistry of the system has allowed rational design of inhibitors of this process, which function as antitumor and antiviral agents.