Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways and many such products have potential as therapeutics. The ATP dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate suggesting recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whilst catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity; it appears to be a conserved strategy in other peptide modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.

Oceanic cyanobacteria are the most abundant oxygen-generating phototrophs on our planet, and therefore, important to life. These organisms are infected by viruses called cyanophages, recently shown to encode metabolic genes that modulate host photosynthesis, phosphorus cycling and nucleotide metabolism. Herein, we report the characterisation of a wild type flavin-dependent viral halogenase (VirX1) from a cyanophage. Notably, halogenases have been previously associated 2 with secondary metabolism, tailoring natural products. Exploration of this viral halogenase reveals it capable of regioselective halogenation of a diverse range of substrates, with a preference for forming aryl iodide species; this has potential implications for the metabolism of the infected host. Until recently, a flavin-dependent halogenase (FDH) capable of iodination in vitro had not been reported. VirX1 is interesting from a biocatalytic perspective showing strikingly broad substrate flexibility, and a clear preference for iodination, as illustrated by kinetic analysis. These factors together render it an attractive tool for synthesis. The selective formation of carbon-halogen (C-X) bonds is of great importance to the pharmaceutical and agrochemical industries 1,2. The introduction of a halogen (X) into a molecule can be used to modulate bioactivity, bioavailability and metabolic stability 1-3. Traditional chemical methodologies of halogenating aromatic substrates generally employ highly reactive reagents and generate harmful waste. As traditional reagents lack components that enable the tuning of product selectivity, they oftentimes generate products in which either only the most nucleophilic position is halogenated or mixtures of products are produced. Conversely, biosynthetic (enzymatic) halogenation is mild, highly selective and utilises simple salts such as NaCl or NH 4 Br as the source of halide while oxygen serves as the oxidant 1-3. Consequently, the discovery and structural characterisation of flavin-dependent halogenases (FDHs) capable of selectively forming C-Cl and C-Br bonds 4,5 and the discovery of a S-adenosylmethionine (SAM)-dependent fluorinase 6 , able to mediate nucleophilic C-F bond formation, attracted considerable attention. Until recently, a FDH capable of generating C-I bonds remained to be discovered and characterised 7 .

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.

Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable, and capable of modulating protein–protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the 25 amino-acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino-acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal eight residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.

Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutic starting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine‐tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger‐scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups, azide or dehydroalanine, that allow chemical diversification, including the use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity, as well as their unnatural analogues, as therapeutics.