Altering glycopeptide antibiotic biosynthesis through mutasynthesis allows incorporation of fluorinated phenylglycine residues.

Glycopeptide antibiotics (GPAs) are peptide natural products used as last resort treatments for antibiotic resistant bacterial infections. They are produced by the sequential activities of a linear nonribosomal peptide synthetase (NRPS), which assembles the heptapeptide core of GPAs, and cytochrome P450 (Oxy) enzymes, which perform a cascade of cyclisation reactions. The GPAs contain proteinogenic and nonproteinogenic amino acids, including phenylglycine residues such as 4-hydroxyphenylglycine (Hpg). The ability to incorporate non-proteinogenic amino acids in such peptides is a distinctive feature of the modular architecture of NRPSs, with each module selecting and incorporating a desired amino acid. Here, we have exploited this ability to produce and characterise GPA derivatives containing fluorinated phenylglycine (F-Phg) residues through a combination of mutasynthesis, biochemical, structural and bioactivity assays. Our data indicate that the incorporation of F-Phg residues is limited by poor acceptance by the NRPS machinery, and that the phenol moiety normally present on Hpg residues is essential to ensure both acceptance by the NRPS and the sequential cyclisation activity of Oxy enzymes. The principles learnt here may prove useful for the future production of GPA derivatives with more favourable properties through mixed feeding mutasynthesis approaches.

An in vitro assay to explore condensation domain specificity from non-ribosomal peptide synthesis.

Non-ribosomal peptide synthesis produces a wide range of bioactive peptide natural products and is reliant on a modular architecture based on repeating catalytic domains able to generate diverse peptide sequences. In this chapter we detail an in vitro biochemical assay to explore the substrate specificity of condensation domains, which are responsible for peptide elongation, from the biosynthetic machinery that produces from the siderophore fuscachelin. This assay removes the requirement to utilise the specificity of adjacent adenylation domains and allows the acceptance of a wide range of synthetic substrates to be explored.

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.

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.

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.

Correction: Novel chemical probes for the investigation of nonribosomal peptide assembly.

Correction for 'Novel chemical probes for the investigation of nonribosomal peptide assembly' by Y. T. Candace Ho et al., Chem. Commun., 2017, 53, 7088-7091.

Novel chemical probes for the investigation of nonribosomal peptide assembly.

Chemical probes were devised and evaluated for the capture of biosynthetic intermediates involved in the bio-assembly of the nonribosomal peptide echinomycin. Putative intermediate peptide species were isolated and characterised, providing fresh insights into pathway substrate flexibility and paving the way for novel chemoenzymatic approaches towards unnatural peptides.