Advancing Nitrile-Aminothiol Strategy for Dual and Sequential Bioconjugation.

Nitrile-aminothiol conjugation (NATC) stands out as a promising biocompatible ligation technique due to its high chemo-selectivity. Herein we investigated the reactivity and substrate scope of NAT conjugation chemistry, thus developing a novel pH dependent orthogonal NATC as a valuable tool for chemical biology. The study of reaction kinetics elucidated that the combination of heteroaromatic nitrile and aminothiol groups led to the formation of an optimal bioorthogonal pairing, which is pH dependent. This pairing system was effectively utilized for sequential and dual conjugation. Subsequently, these rapid (≈1 h) and high yield (>90%) conjugation strategies were successfully applied to a broad range of complex biomolecules, including oligonucleotides, chelates, small molecules and peptides. The effectiveness of this conjugation chemistry was demonstrated by synthesizing a fluorescently labelled antimicrobial peptide-oligonucleotide complex as a dual conjugate to imaging in live cells. This first-of-its-kind sequential NATC approach unveils unprecedented opportunities in modern chemical biology, showcasing exceptional adaptability in rapidly creating structurally complex bioconjugates. Furthermore, the results highlight its potential for versatile applications across fundamental and translational biomedical research.

Reassignment of the Structure of a Tryptophan-Containing Cyclic Tripeptide Produced by the Biarylitide Crosslinking Cytochrome P450blt.

The structure of the sidechain crosslinked Tyr-Leu-Trp peptide produced by the biarylitide crosslinking cytochrome P450Blt from Micromonospora sp. MW-13 has been reanalysed by a series of NMR, computational and isotope labelling experiments and shown to contain a C-N rather than a C-O bond. Additional in vivo experiments using such a modified peptide show there is a general tolerance of biarylitide crosslinking P450 enzymes for histidine to tryptophan mutations within their minimal peptide substrate sequences despite the lack of such residues noted in natural biarylitide gene clusters. This work further highlights the impressive ability of P450s from biarylitide biosynthesis pathways to act as biocatalysts for the formation of a range of sidechain crosslinked tripeptides.

Enzyme engineering lets us play with new building blocks in non-ribosomal peptide synthesis.

In a recent issue of Nature Chemical Biology, Folger et al. demonstrated a high-throughput approach for engineering peptide bond forming domains from non-ribosomal peptide synthesis. A non-ribosomal peptide synthetase module from surfactin biosynthesis was reprogrammed to accept a fatty acid substrate into peptide biosynthesis, thus illustrating the potential of this approach for generating novel bioactive peptides.

An Engineered Biarylitide Cross-Linking P450 from RiPP Biosynthesis Generates Alternative Cyclic Peptides.

Cytochrome-P450-mediated cross-linking of ribosomally encoded peptides (RiPPs) is rapidly expanding and displays great potential for biocatalysis. Here, we demonstrate that active site engineering of the biarylitide cross-linking enzyme P450Blt enables the formation of His-X-Tyr and Tyr-X-Tyr cross-linked peptides, thus showing how such P450s can be further exploited to produce alternate cyclic tripeptides with controlled cross-linking states.

Correction: Synthetic ramoplanin analogues are accessible by effective incorporation of arylglycines in solid-phase peptide synthesis.

[This corrects the article DOI: 10.1039/D3SC01944F.].

Synthetic ramoplanin analogues are accessible by effective incorporation of arylglycines in solid-phase peptide synthesis.

The threat of antimicrobial resistance to antibiotics requires a continual effort to develop alternative treatments. Arylglycines (or phenylglycines) are one of the signature amino acids found in many natural peptide antibiotics, but their propensity for epimerization in solid-phase peptide synthesis (SPPS) has prevented their use in long peptide sequences. We have now identified an optimized protocol that allows the synthesis of challenging non-ribosomal peptides including precursors of the glycopeptide antibiotics and an analogue of feglymycin (1 analogue, 20%). We have exploited this protocol to synthesize analogues of the peptide antibiotic ramoplanin using native chemical ligation/desulfurization (1 analogue, 6.5%) and head-to-tail macrocyclization in excellent yield (6 analogues, 3-9%), with these compounds extensively characterized by NMR (U-shaped structure) and antimicrobial activity assays (two clinical isolates). This method significantly reduces synthesis time (6-9 days) when compared with total syntheses (2-3 months) and enables drug discovery programs to include arylglycines in structure-activity relationship studies and drug development.

Resurrecting ancestral antibiotics: unveiling the origins of modern lipid II targeting glycopeptides.

Antibiotics are central to modern medicine, and yet they are mainly the products of intra and inter-kingdom evolutionary warfare. To understand how nature evolves antibiotics around a common mechanism of action, we investigated the origins of an extremely valuable class of compounds, lipid II targeting glycopeptide antibiotics (GPAs, exemplified by teicoplanin and vancomycin), which are used as last resort for the treatment of antibiotic resistant bacterial infections. Using a molecule-centred approach and computational techniques, we first predicted the nonribosomal peptide synthetase assembly line of paleomycin, the ancestral parent of lipid II targeting GPAs. Subsequently, we employed synthetic biology techniques to produce the predicted peptide and validated its antibiotic activity. We revealed the structure of paleomycin, which enabled us to address how nature morphs a peptide antibiotic scaffold through evolution. In doing so, we obtained temporal snapshots of key selection domains in nonribosomal peptide synthesis during the biosynthetic journey from ancestral, teicoplanin-like GPAs to modern GPAs such as vancomycin. Our study demonstrates the synergy of computational techniques and synthetic biology approaches enabling us to journey back in time, trace the temporal evolution of antibiotics, and revive these ancestral molecules. It also reveals the optimisation strategies nature has applied to evolve modern GPAs, laying the foundation for future efforts to engineer this important class of antimicrobial agents.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,ß-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

Not always an innocent bystander: the impact of stabilised phosphopantetheine moieties when studying nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases produce many important peptide natural products and are centred around carrier proteins (CPs) that deliver intermediates to various catalytic domains. We show that the replacement of CP substrate thioesters by stabilised ester analogues leads to active condensation domain complexes, whereas amide stabilisation generates non-functional complexes.

A Chemoenzymatic Approach to Investigate Cytochrome P450 Cross-Linking in Glycopeptide Antibiotic Biosynthesis.

Glycopeptide antibiotics (GPAs) are important and medically relevant peptide natural products. In the context of antimicrobial resistance (AMR), understanding and manipulating GPA biosynthesis is essential to discover new bioactive derivatives of these peptides. Among all the enzymatic steps in GPA biosynthesis, the most complex occurs during the maturation (cross-linking) of the peptide aglycone. This is achieved-while the peptide remains attached to the nonribosomal peptide synthetase (NRPS) machinery-through the action of a cytochrome P450 (CYP450 or Oxy)-mediated cyclization cascade. There is great interest in understanding the formation of the cross-links between the aromatic side chains in GPAs as this process leads to the cup-shaped aglycone, which is itself a requirement for antibiotic activity. In this regard, the use of in vitro experiments is crucial to study this process. To address the process of peptide cyclization during GPA biosynthesis, a series of peptide substrates and different Oxy enzymes are required. In this chapter, we describe a practical and efficient route for the synthesis of peptidyl-CoAs, the expression of proteins/enzymes involved in the in vitro cyclization assay, the loading of the PCP with peptidyl-CoAs, an optimized CYP450-mediated cyclization cascade and assay workup followed by mass spectrometry (MS) characterization. This in vitro assay affords high conversion to cyclic peptides and demonstrates the tolerance of the P450s for novel GPA precursor peptide substrates.

Metabolic engineering of the shikimate pathway in Amycolatopsis strains for optimized glycopeptide antibiotic production.

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahpsec and Pdhsec) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahpprim and Pdhprim) in the core genome. While overexpression of the dahpsec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdhprim or pdhsec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply.

Chemoselective Methionine Labelling of Recombinant Trastuzumab Shows High In Vitro and In Vivo Tumour Targeting.

A highly effective 2-step system for site-specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine-independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox-activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide-alkyne cycloaddition 'click' conjugation. The versatility of this 2-step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine-modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine-mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.

Exploring the Flexibility of the Glycopeptide Antibiotic Crosslinking Cascade for Extended Peptide Backbones.

The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell-wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II-binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II-binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C-terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C-terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.

Structural diversity, biosynthesis, and biological functions of lipopeptides from Streptomyces.

Covering: up to 2022Streptomyces are ubiquitous in terrestrial and marine environments, where they display a fascinating metabolic diversity. As a result, these bacteria are a prolific source of active natural products. One important class of these natural products is the nonribosomal lipopeptides, which have diverse biological activities and play important roles in the lifestyle of Streptomyces. The importance of this class is highlighted by the use of related antibiotics in the clinic, such as daptomycin (tradename Cubicin). By virtue of recent advances spanning chemistry and biology, significant progress has been made in biosynthetic studies on the lipopeptide antibiotics produced by Streptomyces. This review will serve as a comprehensive guide for researchers working in this multidisciplinary field, providing a summary of recent progress regarding the investigation of lipopeptides from Streptomyces. In particular, we highlight the structures, properties, biosynthetic mechanisms, chemical and chemoenzymatic synthesis, and biological functions of lipopeptides. In addition, the application of genome mining techniques to Streptomyces that have led to the discovery of many novel lipopeptides is discussed, further demonstrating the potential of lipopeptides from Streptomyces for future development in modern medicine.

Beyond vancomycin: recent advances in the modification, reengineering, production and discovery of improved glycopeptide antibiotics to tackle multidrug-resistant bacteria.

Glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin, are important last-resort antibiotics used to treat multidrug-resistant Gram-positive bacterial infections. Whilst second-generation GPAs - generated through chemical modification of natural GPAs - have proven successful, the emergence of GPA resistance has underlined the need to develop new members of this compound class. Significant recent advances have been made in GPA research, including gaining an in-depth understanding of their biosynthesis, improving titre in production strains, developing new derivatives via novel chemical modifications and identifying a new mode of action for structurally diverse type-V GPAs. Taken together, these advances demonstrate significant untapped potential for the further development of GPAs to tackle the growing threat of multidrug-resistant bacteria.

Replacing Commercial 6-Phosphofructokinase in an Online Pyrophosphate Detection Assay.

Detection of pyrophosphate is important in quantifying enzyme activity, particularly adenylation domain activity during non-ribosomal peptide synthesis. The previous development of an enzyme coupled PPi /NADH assay allowed the measurement of such activity in an online fashion using commercially available components. Now, with a key enzyme - 6-phosphofructokinase - no longer available, we have screened and identified viable replacement enzymes that can be expressed in high yield and that are far superior in activity to the now discontinued commercial product. This will support the ability of groups to continue to use this established online assay for pyrophosphate detection.

Cytochrome P450Blt Enables Versatile Peptide Cyclisation to Generate Histidine- and Tyrosine-Containing Crosslinked Tripeptide Building Blocks.

We report our investigation of the utility of peptide crosslinking cytochrome P450 enzymes from biarylitide biosynthesis to generate a range of cyclic tripeptides from simple synthons. The crosslinked tripeptides produced by this P450 include both tyrosine-histidine (A-N-B) and tyrosine-tryptophan (A-O-B) crosslinked tripeptides, the latter a rare example of a phenolic crosslink to an indole moiety. Tripeptides are easily isolated following proteolytic removal of the leader peptide and can incorporate a wide range of amino acids in the residue inside the crosslinked tripeptide. Given the utility of peptide crosslinks in important natural products and the synthetic challenge that these can represent, P450 enzymes have the potential to play roles as important tools in the generation of high-value cyclic tripeptides for incorporation in synthesis, which can be yet further diversified using selective chemical techniques through specific handles contained within these tripeptides.

The Cytochrome P450 OxyA from the Kistamicin Biosynthesis Cyclization Cascade is Highly Sensitive to Oxidative Damage.

Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilize a cysteine thiolate-ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of the active intermediate formed within P450s during their active cycle, it is remarkable that these enzymes can avoid auto-oxidation and retain the axial cysteine ligand in the deprotonated-and thus highly acidic-thiolate form. While little is known about the process of heme incorporation during P450 folding, there is an overwhelming preference for one heme orientation within the P450 active site. Indeed, very few structures to date contain an alternate heme orientation, of which two are OxyA homologs from glycopeptide antibiotic (GPA) biosynthesis. Given the apparent preference for the unusual heme orientation shown by OxyA enzymes, we investigated the OxyA homolog from kistamicin biosynthesis (OxyAkis), which is an atypical GPA. We determined that OxyAkis is highly sensitive to oxidative damage by peroxide, with both UV and EPR measurements showing rapid bleaching of the heme signal. We determined the structure of OxyAkis and found a mixed population of heme orientations present in this enzyme. Our analysis further revealed the possible modification of the heme moiety, which was only present in samples where the alternate heme orientation was present in the protein. These results suggest that the typical heme orientation in cytochrome P450s can help prevent potential damage to the heme-and hence deactivation of the enzyme-during P450 catalysis. It also suggests that some P450 enzymes involved in GPA biosynthesis may be especially prone to oxidative damage due to the heme orientation found in their active sites.

Antibiotic-chemoattractants enhance neutrophil clearance of Staphylococcus aureus.

The pathogen Staphylococcus aureus can readily develop antibiotic resistance and evade the human immune system, which is associated with reduced levels of neutrophil recruitment. Here, we present a class of antibacterial peptides with potential to act both as antibiotics and as neutrophil chemoattractants. The compounds, which we term 'antibiotic-chemoattractants', consist of a formylated peptide (known to act as chemoattractant for neutrophil recruitment) that is covalently linked to the antibiotic vancomycin (known to bind to the bacterial cell wall). We use a combination of in vitro assays, cellular assays, infection-on-a-chip and in vivo mouse models to show that the compounds improve the recruitment, engulfment and killing of S. aureus by neutrophils. Furthermore, optimizing the formyl peptide sequence can enhance neutrophil activity through differential activation of formyl peptide receptors. Thus, we propose antibiotic-chemoattractants as an alternate approach for antibiotic development.