Leader peptide exchange to produce hybrid, new-to-nature ribosomal natural products

Ribosomal natural products contain exquisite post-translational peptide modifications that are installed by a range of pathway-specific enzymes. We present proof of principle for a Sortase A-based approach that enables peptide modifcation by enzymes from unrelated pathways. This allowed the one-pot synthesis of a new-to-nature, hybrid ribosomal natural product

Facile Production of the Pseudomonas aeruginosa Virulence Factor LasB in Escherichia coli for Structure-Based Drug Design

The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor

Facile production of the Pseudomonas aeruginosa virulence factor LasB in Escherichia coli for structure‐based drug design

The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high‐level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor

Thiazoline-specific amidohydrolase PurAH is the gatekeeper of bottromycin biosynthesis

The ribosomally synthesized and post-translationally modified peptide (RiPP) bottromycin A2 possesses potent antimicrobial activity. Its biosynthesis involves the enzymatic formation of a macroamidine, a process previously suggested to require the concerted efforts of a YcaO enzyme (PurCD) and an amidohydrolase (PurAH) in vivo. In vitro, PurCD alone is sufficient to catalyze formation of the macroamidine, but the process is reversible. We set out to probe the role of PurAH in macroamidine formation in vitro. We demonstrate that PurAH is highly selective for macroamidine-containing precursor peptides and cleaves C-terminal of a thiazoline, thus removing the follower peptide. After follower cleavage, macroamidine formation is irreversible, indicating PurAH as the gatekeeper of bottromycin biosynthesis. The structure of PurAH suggests residues involved in catalysis, which were probed through mutagenesis

The structural biology of patellamide biosynthesis

This work was supported by grants from the ERC339367 (JHN and MJ) and BBSRCBB/K015508/1 (JHN and MJ).The biosynthetic pathways for patellamide and related natural products have recently been studied by structural biology. These pathways produce molecules that have a complex framework and exhibit a diverse array of activity due to the variability of the amino acids that are found in them. As these molecules are difficult to synthesize chemically, exploitation of their properties has been modest. The patellamide pathway involves amino acid heterocyclization, peptide cleavage, peptide macrocyclization, heterocycle oxidation and epimerization; closely related products are also prenylated. Enzyme activities have been identified for all these transformations except epimerization, which may be spontaneous. This review highlights the recent structural and mechanistic work on amino acid heterocyclization, peptide cleavage and peptide macrocyclization. This work should help in using the enzymes to produce novel analogs of the natural products enabling an exploitation of their properties.Peer reviewe

Bottromycins - biosynthesis, synthesis and activity

Covering: 1950s up to the end of 2020Bottromycins are a class of macrocyclic peptide natural products that are produced by several Streptomyces species and possess promising antibacterial activity against clinically relevant multidrug-resistant pathogens. They belong to the ribosomally synthesised and post-translationally modified peptide (RiPP) superfamily of natural products. The structure contains a unique four-amino acid macrocycle formed via a rare amidine linkage, C-methylation and a d-amino acid. This review covers all aspects of bottromycin research with a focus on recent years (2009-2020), in which major advances in total synthesis and understanding of bottromycin biosynthesis were achieved

Structure and substrate recognition of the Bottromycin maturation enzyme BotP

JK would like to thank the University of St Andrews, which is supported by a Wellcome Trust Capital Award (086036) and the Deutsche Forschungsgemeinschaft for an Emmy Noether fellowship (KO4116/3-1). BN would like to thank the European Research Council (339367).The bottromycins are a family of highly modified peptide natural products displaying potent antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphyloccoccus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N- terminal "leader" sequence. We report the structural and biochemical characterization of BotP, a leucyl-aminopeptidase like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of apo BotP and of BotP in complex with Mn2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottromycin.PostprintPeer reviewe

Photorhabdus luminescens lectin A (PllA) : A new probe for detecting α-galactoside-terminating glycoconjugates

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for -galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa.We also investigated the utility of PllA as a probe for detecting -galactosides. The -Gal epitope is present on wildtype pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA’s high specificity for -galactoside–containing ligands, and we show that PllA can be used to visualize the -Gal epitope on porcine tissues

Structure of PatF from Prochloron didemni

Patellamides are macrocyclic peptides with potent biological effects and are a subset of the cyanobactins. Cyanobactins are natural products that are produced by a series of enzymatic transformations and a common modification is the addition of a prenyl group. Puzzlingly, the pathway for patellamides in Prochloron didemni contains a gene, patF, with homology to prenylases, but patellamides are not themselves prenylated. The structure of the protein PatF was cloned, expressed, purified and determined. Prenylase activity could not be demonstrated for the protein, and examination of the structure revealed changes in side-chain identity at the active site. It is suggested that these changes have inactivated the protein. Attempts to mutate these residues led to unfolded protein

Trapping of a polyketide synthase module after C−C bond formation reveals transient acyl carrier domain interactions

Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)‐bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter‐domain interactions after C−C bond formation in a chain‐branching module of the rhizoxin PKS. Mechanism‐based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C−C bond formation by cryo‐electron microscopy (cryo‐EM). The possible existence of two ACP binding sites, one of them a potential “parking position” for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C−C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines