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 as biocatalysts for the formation of a range of sidechain crosslinked tripeptides.

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.

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.

Further Developments towards a Minimal Potent Derivative of Human Relaxin-2.

Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1; however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.

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.

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.

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.

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.

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics.

The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules (M) 1-4 of the NRP synthetase (NRPS) assembly lines that synthesise the clinically relevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPS-mediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)-amino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems.

Understanding the Glycopeptide Antibiotic Crosslinking Cascade: In Vitro Approaches Reveal the Details of a Complex Biosynthesis Pathway.

The glycopeptide antibiotics (GPAs) are a fascinating example of complex natural product biosynthesis, with the nonribosomal synthesis of the peptide core coupled to a cytochrome P450-mediated cyclisation cascade that crosslinks aromatic side chains within this peptide. Given that the challenges associated with the synthesis of GPAs stems from their highly crosslinked structure, there is great interest in understanding how biosynthesis accomplishes this challenging set of transformations. In this regard, the use of in vitro experiments has delivered important insights into this process, including the identification of the unique role of the X-domain as a platform for P450 recruitment. In this minireview, we present an analysis of the results of in vitro studies into the GPA cyclisation cascade that have demonstrated both the tolerances and limitations of this process for modified substrates, and in turn developed rules for the future reengineering of this important antibiotic class.

Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.

The receptor for the neuropeptide relaxin 3, relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure-activity relationship studies on both human relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms.

A Chemoenzymatic Approach to the Synthesis of Glycopeptide Antibiotic Analogues.

Glycopeptide antibiotics (GPAs) are important antibiotics that are highly challenging to synthesise due to their unique and heavily crosslinked structure. Given this, the synthetic production and diversification of this key compound class remains impractical. Furthermore, the possibility of biosynthetic reengineering of GPAs is not yet feasible since the selectivity of the biosynthetic crosslinking enzymes for altered substrates is largely unknown. We show that combining peptide synthesis with enzymatic cyclisation enables the formation of novel examples of GPAs and provides an indication of the utility of these crucial enzymes. By accessing the biosynthetic process in vitro, we identified peptide modifications that are enzymatically tolerated and can also reveal the mechanistic basis for substrate intolerance where present. Using this approach, we next specifically activated modified residues within GPAs for functionalisation at previously inaccessible positions, thereby offering the possibility of late-stage chemical functionalisation after GPA cyclisation is complete.

Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase.

Non-ribosomal peptide synthesis is an important biosynthesis pathway in secondary metabolism. In this study we have investigated modularisation and redesign strategies for the glycopeptide antibiotic teicoplanin. Using the relocation or exchange of domains within the NRPS modules, we have identified how to initiate peptide biosynthesis and explored the requirements for the functional reengineering of both the condensation/adenylation domain and epimerisation/condensation domain interfaces. We have also demonstrated strategies that ensure communication between isolated NRPS modules, leading to new peptide assembly pathways. This provides important insights into NRPS reengineering of glycopeptide antibiotic biosynthesis and has broad implications for the redesign of other NRPS systems.

Exploring the Tetracyclization of Teicoplanin Precursor Peptides through Chemoenzymatic Synthesis.

The glycopeptide antibiotics (GPAs) serve as an important example of the interplay of two powerful enzymatic classes in secondary metabolism: the coupling of nonribosomal peptide synthesis with oxidative aromatic cross-linking performed by cytochrome P450 enzymes. This interplay is responsible for the generation of the highly cross-linked peptide aglycone at the core of this compound class that is required for antibiotic activity and, as such, serves as an important point for the exploration of chemoenzymatic routes to understand the selectivity and mechanism of this complex cascade. Here, we demonstrate the effective reconstitution of enzymatic tetracyclization of synthetic teicoplanin-derived heptapeptides and furthermore discern the importance of the OxyE enzyme in maintaining effective cyclization of such peptides bearing 3,5-dihydroxyphenylglycine residues at position 3 in their structures. These results demonstrate the value of chemically synthesized probes for the elucidation of the enzyme mechanism underpinning the complex process of GPA cyclization and furthermore show the utility of the technique for probing the cyclization of non-natural GPA peptides by these powerful biosynthetic enzymes.

Biological, chemical, and biochemical strategies for modifying glycopeptide antibiotics.

Since the discovery of vancomycin in the 1950s, the glycopeptide antibiotics (GPAs) have been of great interest to the scientific community. These nonribosomally biosynthesized peptides are highly cross-linked, often glycosylated, and inhibit bacterial cell wall assembly by interfering with peptidoglycan synthesis. Interest in glycopeptide antibiotics covers many scientific disciplines, due to their challenging total syntheses, complex biosynthesis pathways, mechanism of action, and high potency. After intense efforts, early enthusiasm has given way to a recognition of the challenges in chemically synthesizing GPAs and of the effort needed to study and modify GPA-producing strains to prepare new GPAs to address the increasing threat of microbial antibiotic resistance. Although the preparation of GPAs, either by modifying the pendant groups such as saccharides or by functionalizing the N- or C-terminal moieties, is readily achievable, the peptide core of these molecules-the GPA aglycone-remains highly challenging to modify. This review aims to present a summary of the results of GPA modification obtained with the three major approaches developed to date: in vivo strain manipulation, total chemical synthesis, and chemoenzymatic synthesis methods.

Enzymatic Cascade To Evaluate the Tricyclization of Glycopeptide Antibiotic Precursor Peptides as a Prequel to Biosynthetic Redesign.

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.