Biology subjects

Challis, G. L.

Publications and source records attributed to Challis, G. L..

12 recordsLinked to original sources

bioRxiv2026

Cryptic starter amidation in antibiotic biosynthesis by trans-acyltransferase polyketide synthases

Polyketide biosynthesis is typically initiated by loading a starter unit onto an acyl carrier protein (ACP). In type I modular polyketide synthases (PKSs), responsible for the assembly of diverse bioactive metabolites in bacteria, this ACP is usually incorporated into a chain initiation module, alongside a starter unit loading domain. In several cases, the starter unit undergoes structural modification prior to the initiation of chain assembly. Gladiolin, an antibiotic with promising activity against bacterial and fungal pathogens, is assembled by a trans-acyltransferase (AT) PKS in Burkholderia gladioli. It appears to incorporate a succinyl starter unit, but the gladiolin PKS lacks a conventional loading module, making it unclear how this happens. The gladiolin biosynthetic gene cluster encodes an AT of unassigned function (GbnB), an ACP (GbnA), and an asparagine synthetase homolog (GbnC) with similarity to enzymes that amidate the malonyl-ACP starter unit in glutarimide antibiotic biosynthesis. Here, we elucidate a cryptic starter unit amidation mechanism in gladiolin biosynthesis involving these three proteins. GbnB loads a succinyl unit onto the phosphopantetheinyl arm of GbnA, which is subsequently amidated by GbnC using glutamine as the nitrogen donor. After the fully assembled polyketide chain is released, GbnM hydrolyzes the amide, yielding mature gladiolin. Phylogenetic analyses, coupled with gene cluster reannotation revealed analogous enzymatic machinery likely responsible for cryptic succinamyl and malonamyl starter unit incorporation into etnangien and sorangicin A, respectively. Retro-biosynthetic analyses suggest succinamyl and malonamyl starter units may be involved in the assembly of other metabolites, such as the sorangiolides and azumamides.

biochemistry↗

bioRxiv2025

Substrate scope and catalytic mechanism of α, β-epoxyketone synthase EpnF illuminated by in-situ esterase-mediated deprotection

, {beta}-Epoxyketones are an important class of bacterial natural products with wide-ranging potential applications in oncology, immunology, and infectious disease. Their clinical potential derives from the , {beta}-epoxyketone pharmacophore, which covalently modifies the N-terminal catalytic threonine residue of proteasome {beta}-subunits with high selectivity. Although a synthetic , {beta}-epoxyketone (Carfilzomib) is approved for clinical use, stereo-controlled synthesis of the pharmacophore remains challenging, involving either multiple steps and energy-intensive processes, or unsustainable reagents. Unusual flavoenzymes catalyze the assembly of the pharmacophore in , {beta}-epoxyketone biosynthesis. A detailed understanding of the substrate scope and catalytic mechanism of these enzymes has thus far been limited by the intrinsic instability of their - (di)methyl-{beta}-ketoacid substrates. Here, we report the development and application of an esterase-mediated unmasking strategy for in-situ generation of these substrates from the corresponding methyl esters. Using this approach, we demonstrate that EpnF, the epoxyketone synthase involved in eponemycin / TMC-86A biosynthesis, tolerates a broad range of synthetic substrate analogs, including several with N-terminal protecting groups widely used in peptide synthesis. These findings establish that EpnF has the potential to be developed into a useful biocatalyst for the chemoenzymatic synthesis of dipeptidyl epoxyketone precursors of clinically approved drugs and drug candidates. To elucidate the molecular basis for catalysis of , {beta}-epoxyketone formation by EpnF, substrate docking and molecular dynamics simulations were performed on a well-validated AlphaFold model, providing support for a previously proposed decarboxylation-dehydrogenation-monooxygenation mechanism. Site-directed mutagenesis and LC-MS analysis validated the proposed roles of key active-site residues in substrate positioning and catalysis of epoxide formation. Collectively, these results demonstrate that EpnF and related enzymes belong to a new class of internal flavoprotein monooxygenases and provide a foundation for developing epoxyketone synthases into useful biocatalysts for the sustainable synthesis of high-value ,{beta}-epoxyketones.

biochemistry↗

bioRxiv2025

Bioinformatics-based stereochemical elucidation of MM 46115: an unusual antiviral spirotetronate that inhibits clathrin-mediated endocytosis

The influenza virus is a leading cause of respiratory tract infection in humans, with the emergence of antiviral drug resistance posing an ongoing challenge to the treatment of influenza. This underscores the urgent need for effective new antiviral treatments. Spirotetronates are a family of bacterial natural products with a range of potent bioactivities. Some members of this family have antiviral activity. Here, we report the bioinformatics-based stereochemical assignment, antiviral properties, and mechanism of action of the unusual type II spirotetronate MM 46115 (renamed pellemicin). We show that pellemicin is active against both influenza A and B viruses at non-toxic concentrations, and inhibits clathrin-mediated endocytosis, which is required for virus internalisation. Given the need for new treatments for viral infections, the results of our work suggest that pellemicin and related spirotetronates could provide a basis for developing promising alternatives to existing antivirals to combat drug-resistant influenza. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/677008v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@177aacforg.highwire.dtl.DTLVardef@16f0b26org.highwire.dtl.DTLVardef@144f9dcorg.highwire.dtl.DTLVardef@16fe673_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMM 46115 (here renamed pellemicin) produced by a mycetoma pathogen is a promising antiviral C_LIO_LIThe pellemicin biosynthetic gene cluster was identified in Actinomadura pelletieri DSM 43383 C_LIO_LISequence analysis of biosynthetic enzymes enabled full stereochemical assignment C_LIO_LIPellemicin inhibits clathrin-mediated endocytosis, an early stage of the viral life cycle C_LI SignificanceThe influenza virus is a respiratory pathogen with epidemic and pandemic potential, resulting in tens of thousands of deaths each year and causing significant health, social and economic impact. The rise of resistant influenza strains against existing antiviral drugs creates a pressing need for alternative treatments. Natural products - specialised metabolites of bacteria, fungi, and plants that often have antimicrobial activity - are a promising source of therapeutics to treat human disease. Here, we identify a cryptic polyketide biosynthetic gene cluster in the genome sequence of Actinomadura pelletieri DSM 43383, an Actinomycete that causes mycetoma in humans. This is proposed to direct production of the previously reported spirotetronate MM 46115 (here renamed pellemicin), which has activity against the influenza virus. Sequence analyses of a polyketide synthase and Diels-Alderase involved in pellemicin biosynthesis, combined with comparative analysis of NMR data for other spirotetronates, enabled us to fully resolve longstanding stereochemical ambiguities in the structure of pellemicin. This underscores the emerging power of bioinformatics-based approaches, and their complementarity to traditional spectroscopic methods, for natural product structure elucidation. We also report extensive insights into the antiviral properties and mechanism of pellemicin, demonstrating that it exhibits antiviral activity at non-toxic doses for up to eight hours after administration and inhibits clathrin-mediated endocytosis, a process that many viruses exploit to enter cells. Only one other bacterial natural product, ikarugamycin, is known to inhibit clathrin-mediated endocytosis. Ikarugamycin contains a tetramate group, which bears a close structural relationship to the tetronate moiety of pellemicin, suggesting that inhibition of clathrin-mediated endocytosis may be a general mechanism of action for antiviral spirotetronates. Our findings therefore indicate that pellemicin, and related spirotetronate natural products, could form a basis for development of new drugs to combat antiviral resistance, and serve as useful tools to study the process of endocytosis.

microbiology↗

bioRxiv2023

Antibiotic skeletal diversification via differential enoylreductase recruitment and module iteration in trans-acyltransferase polyketide synthases

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the "blind watchmaker" of evolution creates skeletal diversity is a key question in contemporary natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug resistant Mycobacterium tuberculosis, and entangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons.

biochemistry↗

bioRxiv2023

Molecular basis for short-chain thioester hydrolysis by acyl hydrolase domains in trans-acyltransferase polyketide synthases

Polyketide synthases (PKSs) are multi-domain enzymatic assembly lines that biosynthesise a wide selection of bioactive natural products from simple building blocks. In contrast to their cis-acyltransferase (AT) counterparts, trans-AT PKSs rely on stand-alone AT domains to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery. Trans-AT PKS gene clusters also encode acyl hydrolase (AH) domains, which are predicted to share the overall fold of AT domains, but hydrolyse aberrant acyl chains from ACP domains, thus ensuring efficient polyketide biosynthesis. How such domains specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH domain and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AH and ACP domains contributes to recognition of cognate and non-cognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the production of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis by trans-AT PKSs, and provide foundations for future bioengineering efforts.

biochemistry↗

bioRxiv2023

Metagenomic domain substitution for the high-throughput modification of non-ribosomal peptide analogues

Non-ribosomal peptides are a diverse and medically important group of natural products. They are biosynthesised by modular non-ribosomal peptide synthetase (NRPS) assembly-lines in which domains from each module act in concert to incorporate a specific amino acid into a peptide. This modular biosynthesis has driven efforts to generate new peptide analogues by substituting amino acid specifying domains. Rational NRPS engineering has increasingly focused on using evolutionarily favoured recombination sites for domain substitution. Here, we present an alternative approach inspired by evolution, which involves large-scale diversification and screening. By adopting a metagenomic approach of amplifying amino acid specifying domains from metagenomic DNA derived from soil, we were able to substitute over 1,000 unique domains into a pyoverdine NRPS. To identify functional domain substitutions, we employed fluorescence and mass spectrometry screening techniques, followed by sequencing. This comprehensive screening process successfully identified more than 100 functional domain substitutions, resulting in the production of 16 distinct pyoverdines as major products. The significance of this metagenomic approach lies in its ability to shift the focus of engineering non-ribosomal peptide biosynthesis. Instead of relying on a high success rate of individual domain substitution, we have developed effective methods that enable the exploration of a broader range of substitutions. This opens new possibilities for the discovery and production of novel non-ribosomal peptides with diverse biological activities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/543161v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@8ed078org.highwire.dtl.DTLVardef@188a0eorg.highwire.dtl.DTLVardef@10fd38corg.highwire.dtl.DTLVardef@9c651_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active against Multi-drug resistant Tuberculosis

Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, in particular their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinobacterial extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2475 and Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus (MRSA) and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 M. In vitro translation assays showed that, like other thiopeptide antibiotics, persiathiacin A targets protein biosynthesis. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster. A cytochrome P450 encoded by this gene cluster catalyses the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyse the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. The discovery of the persiathiacins and their biosynthetic gene cluster thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.

microbiology↗

bioRxiv2021

Exploration of polyyne biosynthetic gene cluster diversity in bacteria leads to the discovery of the Pseudomonas polyyne protegencin

Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources. In bacteria their biosynthesis has been defined, however, the distribution of polyyne biosynthetic gene clusters (BGCs), and their evolutionary relationship to alkyne biosynthesis, have not been addressed. We explored the distribution of alkyne biosynthesis gene cassettes throughout bacteria, revealing evidence of multiple horizontal gene transfer events. Investigating the evolutionary connection between alkyne and polyyne biosynthesis identified a monophyletic clade possessing a conserved seven-gene cassette for polyyne biosynthesis. Mapping the diversity of these conserved genes revealed a phylogenetic clade representing a polyyne BGC in Pseudomonas, pgn, and subsequent pathway mutagenesis and analytical chemistry characterised the associated metabolite, protegencin. In addition to unifying and expanding our knowledge of polyyne diversity, our results show that alkyne and polyyne biosynthetic gene clusters are promiscuous within bacteria. Systematic mapping of conserved biosynthetic genes across bacterial genomic diversity has proven to be a successful method for discovering natural products.

microbiology↗

bioRxiv2020

Structural basis for control of antibiotic production by bacterial hormones

Actinobacteria produce numerous antibiotics and other specialised metabolites with important applications in medicine and agriculture. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR. Here, we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding by single particle cryo-electron microscopy of an MmfR-operator complex. Electrophoretic mobility shift assays with MmfR mutants and synthetic AHFCA analogues illuminate the role played by individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of Actinobacterial hormones and their associated TFTRs in synthetic biology and novel antibiotic discovery.

biochemistry↗

bioRxiv2020

Kill and cure: genomic phylogeny and bioactivity of a diverse collection of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles

Burkholderia gladioli is one of few bacteria with a broad ecology spanning disease in humans, animals, and plants, and encompassing beneficial interactions with multiple eukaryotic hosts. It is a plant pathogen, a bongkrekic acid toxin producing food-poisoning agent, and a lung pathogen in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the ecological diversity and specialized metabolite biosynthesis of 206 B. gladioli strains, phylogenomically defining 5 evolutionary clades. Historical disease pathovars (pv) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were phylogenetically distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable. Soft-rot disease and CF infection pathogenicity traits were conserved across all pathovars. Biosynthetic gene clusters for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade specific. Strikingly, 13% of CF-infection strains characterised (n=194) were bongkrekic acid toxin positive, uniquely linking this food-poisoning risk factor to chronic lung disease. Toxin production was suppressed by exposing strains to the antibiotic trimethoprim, providing a potential therapeutic strategy to minimise poisoning risk in CF.

microbiology↗

bioRxiv2019

Pentamycin biosynthesis in Philippine Streptomyces sp. S816: Cytochrome P450-catalysed installation of the C-14 hydroxyl group

Pentamycin is a polyene antibiotic, registered in Switzerland for the treatment of vaginal candidiasis, trichomo-niasis and mixed infections. Chemical instability has hindered its wide-spread application and development as a drug. Here we report the identification of Streptomyces sp. S816, isolated from Philippine mangrove soil, as a pentamycin producer. Genome sequence analysis identified the putative pentamycin biosynthetic gene cluster, which shows a high degree of similarity to the gene cluster responsible for filipin III biosynthesis. The ptnJ gene, which is absent from the filipin III biosynthetic gene cluster, was shown to encode a cytochrome P450 capable of converting filipin III to pentamycin. This confirms that the cluster directs pentamycin biosynthesis, paving the way for biosynthetic engineering approaches to the production of pentamycin analogues. Several other Streptomyces genomes were found to contain ptnJ orthologues clustered with genes encoding polyketide synthases that appear to have similar architectures to those responsible for the assembly of filipin III and pentamycin, suggesting pentamycin production may be common in Streptomyces species.

biochemistry↗

bioRxiv2017

clusterTools: Proximity Searches For Functional Elements To Identify Putative Biosynthetic Gene Clusters

Motivation: The low cost of DNA sequencing has accelerated research in natural product biosynthesis allowing us to rapidly link small molecules to the clusters that produce them. However, the large amount of data means that the number of putative biosynthetic gene clusters (BGCs) far exceeds our ability to experimentally characterize them. This necessitates the need for development of further tools to analyze putative BGCs to flag those of interest for further characterization.\n\nResults: Clustertools implements a framework to aid in the characterization of putative BGCs. It does this by or-ganizing genomic information on coding sequences in a way that enables directed, hypothesis-driven queries for functional elements in close physical proximity of each other. Genomic sequence databases can be constructed in clusterTools with an interface to the NCBI Genbank and Genomes databases, or from private sequence databases. clusterTools can be used either to identify interesting BGCs from a database of putative BGCs, or on databases of genomic sequences to identify and download regions of interest in the DNA for further processing and annotation in programs such as antiSMASH. We have used clusterTools to identify putative and known biosynthetic gene clus-ters involved in bacterial polyketide alkaoloid and tetronate biosynthesis.\n\nAvailability and Implementation: Clustertools is implemented in Python and is available via the AGPL. Stand-alone versions of clusterTools are available for Macintosh, Windows, and Linux upon registration (https://goo.gl/forms/QRKTkpqiA0g31IWp1). The source-code is available at https://www.github.com/emzodls/clusterArch.\n\nSupplementary information: A manual describing the Python toolkit that powers clusterTools, as well as the HMMs constructed for the tetronate search is available online.

bioinformatics↗