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Macrocyclisation of fully non-peptidic compounds and non-heterocycle containing macrocycles using the peptidic ligase PatGmac., Biocatalysis is a fast developing field in which an enzyme's natural capabilities are harnessed or engineered for synthetic chemistry. The enzyme PatG is an extremely promiscuous macrocyclase enzyme tolerating both non-natural amino acids and non-amino acids within the substrate. It does, however, require a proline or thiazoline at the C-terminal position of the core peptide which means the final product must contain this group. Here, we show guided by structural insight we have identified two synthetic routes, triazole and a double cysteine, that circumvent this requirement. With the triazole, we show PatGmac can macrocyclise substrates that do not contain any amino acids in the final product.

The peptide bond, the defining feature of proteins, governs peptide chemistry by abolishing nucleophilicity of the nitrogen. This and the planarity of the peptide bond arise from the delocalization of the lone pair of electrons on the nitrogen atom into the adjacent carbonyl. While chemical methylation of an amide bond uses a strong base to generate the imidate, OphA, the precursor protein of the fungal peptide macrocycle omphalotin A, self-hypermethylates amides at pH 7 using S-adenosyl methionine (SAM) as cofactor. The structure of OphA reveals a complex catenane-like arrangement in which the peptide substrate is clamped with its amide nitrogen aligned for nucleophilic attack on the methyl group of SAM. Biochemical data and computational modeling suggest a base-catalyzed reaction with the protein stabilizing the reaction intermediate. Backbone N-methylation of peptides enhances their protease resistance and membrane permeability, a property that holds promise for applications to medicinal chemistry., Science Advances, 4 (8), ISSN:2375-2548

This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1), the Wellcome Trust (TripleTOF 5600 mass spectrometer (094476), the MALDI TOF-TOF Analyser (079272AIA), 700 NMR) and the EPSRC UK National Mass Spectrometry Facility at Swansea University. J.H.N. is a Royal Society Wolfson Merit Award Holder and 1000 talent scholar at Sichuan University Biocatalysis is a fast developing field in which an enzyme’s natural capabilities are harnessed or engineered for synthetic chemistry. The enzyme PatG is an extremely promiscuous macrocyclase enzyme tolerating both non-natural amino acids and non-amino acids within the substrate. It does, however, require a proline or thiazoline at the C-terminal position of the core peptide which means the final product must contain this group. Here, we show guided by structural insight we have identified two synthetic routes, triazole and a double cysteine, that circumvent this requirement. With the triazole, we show PatGmac can macrocyclise substrates that do not contain any amino acids in the final product. Publisher PDF

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3'-deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5'-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3'-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.

This work was supported by funds from the St Andrews EM facility and the University of St Andrews. CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government. CP is also supported by a Tenovus Scotland Award (T15/41). JML was supported by a Programme grant from the Wellcome Trust (Number 045404). Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures. Publisher PDF

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Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and co-factors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that AMP is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Regulation of Kef system function is via the reversible binding of comparatively low affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilised, at least in part, by AMP binding.

This thesis reports on the structural and functional studies performed using x-ray crystallography on two proteins involved in bacterial sugar metabolism. The first protein, the inner membrane oligosaccharide scramblase Wzx, is central to capsule and lipopolysaccharide export in pathogens and is thus a potential drug target. The crystal structure was determined at 2.3 Å using sulfur phasing at the long wavelength beamline I23 at Diamond Light Source. This required us to obtain high quality crystals using lipidic cubic phase, exogenous lipids, and a nanobody. The crystal structure reveals a positively charged patch at both openings to a central cavity. The structure will guide future site directed mutagenesis and mechanistic studies. Other nanobodies were identified and these could help trap other conformational states. The second protein studied was the sialic acid oxidoreductase RgNanOx. This protein used in sialic acid metabolism by the gut bacterium Ruminococcus gnavus, which converts sialic acid (Neu5Ac) into the bicyclic form (2,7-anhydro-Neu5Ac). This involves an unusual chemical mechanism where the substrate is both oxidised and reduced. The crystal structure of RgNanOx was first determined at 1.7 Å. The structure suggested a mechanism for this unusual chemical transformation which was tested by site directed mutagenesis. The enzyme was shown to be active against the sialidase inhibitor 2,3-dehydro-2-deoxy-Neu5Ac (DANA). Co-crystallisation with DANA produced an α-Neu5Ac complex. A mutant was used to obtain a 2,7-anhydro-Neu5Ac complex. These complexes allow the mechanism to be clarified and the key catalytic residues identified. It is an unresolved question whether the true substrate is the α or β form of the sialic acid. NMR data suggest that α-Neu5Ac is consumed, which would indicate the enzyme proceeds by an unusual syn- elimination. The work opens opportunities to definitively assign the mechanism, and shed light on the role of the enzyme in the gut environment.