Biology subjects

Jenner, M.

Publications and source records attributed to Jenner, M..

11 recordsLinked to original sources

bioRxiv2026

Structural and biochemical characterisation of an iterative GCN5-related N-acetyltransferase required for fungal siderophore tailoring

Siderophore-mediated iron acquisition is essential for fungal survival, particularly under iron-limiting conditions. In Aspergillus fumigatus, SidG, a member of the GCN5-related N-acetyltransferase (GNAT) superfamily, catalyses the final step in the biosynthesis of the extracellular siderophore triacetylfusarinine C (TAFC) through sequential acetylation of the precursor fusarinine C (FsC). However, the timing, catalytic mechanism, and functional significance of this modification are not fully understood. Here, we reconstituted SidG activity in vitro and combined native mass spectrometry, X-ray crystallography, molecular dynamics simulations, and site-directed mutagenesis to investigate its catalytic properties. Our analyses demonstrate that SidG selectively binds acetyl-CoA from the cellular milieu and iteratively acetylates the FsC scaffold prior to iron chelation. Structural, biochemical, and molecular dynamics analyses support a direct transfer mechanism, identify key catalytic residues, and demonstrate the strict selectivity of SidG for short-chain acyl-CoA donors. Together, these findings establish the molecular basis for SidG-dependent siderophore tailoring and expand our understanding of GNAT-catalysed transformations in fungal natural product biosynthesis.

biochemistry↗

bioRxiv2025

Molecular characterisation of the acyltransferase-acyl carrier protein interface in a fungal highly reducing polyketide synthase.

Iterative polyketide synthases (iPKSs) rely on communication between acyl carrier protein (ACP) and acyltransferase (AT) domains to ensure efficient delivery of starter and extender substrates during biosynthesis. However, the molecular determinants governing the AT:ACP interface remain poorly understood. Here, we use the fungal highly reducing PKS, SimG, a component of the cyclosporin biosynthetic pathway, as a model system to dissect the AT:ACP interface. Using alanine scanning mutagenesis combined with a high-throughput intact-protein mass spectrometry assay, we identified epitope-forming residues that affect AT:ACP interaction. These experimental constraints were used to guide docking and molecular dynamics simulations to produce a data-driven structural model of the SimG AT:ACP complex in a catalytically competent geometry. We also demonstrate that the SimG AT domain transacylates ACP domains from a range of fungal PKS architectural classes, highlighting significant interface plasticity. These insights advance our fundamental understanding of domain communication in these enigmatic megasynthases and provide a foundation for rational engineering to expand substrate scope towards novel polyketide scaffolds.

biochemistry↗

bioRxiv2025

Molecular basis for depsipeptide HDAC inhibitor combinatorial biosynthesis

Polyketides and nonribosomal peptides are important natural product classes with wide-ranging medical and agricultural applications. The analogous enzymatic logic employed by bacterial modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) enables the assembly of hybrid products. One important group of polyketide-nonribosomal peptide hybrids is exemplified by the HDAC-targeting drug romidepsin. This group is assembled by combinatorial biosynthesis involving fusion of a conserved Zn2+-binding pharmacophore to a variable peptide-based cap. Here, we use gene proximity searching to identify the FR-901375 biosynthetic gene cluster in Pseudomonas chlororaphis subsp. piscium DSM 21509. Comparison of the PKS-NRPS encoded by this gene cluster with those assembling related depsipeptide HDAC inhibitors suggests a novel subunit docking modality enables interaction between the conserved pharmacophore and variable cap biosynthetic machineries. This hypothesis was validated using crosstalk assays, mutagenesis, AlphaFold predictions, and carbene footprinting, providing new insight into the evolution of mechanisms for hybrid polyketide-nonribosomal peptide combinatorial biosynthesis.

biochemistry↗

bioRxiv2025

PARAS: high-accuracy machine-learning of substrate specificities in nonribosomal peptide synthetases

Nonribosomal peptides are diverse natural products with important applications in medicine and agriculture. Bacterial and fungal genomes contain thousands of nonribosomal peptide biosynthetic gene clusters (BGCs) of unknown function, providing a promising resource for peptide discovery. Core structural features of such peptides can be inferred by predicting the substrate(s) of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). However, existing approaches to A domain prediction rely on limited datasets and often struggle with domains selecting large substrates or from less-studied taxa. Here, we systematically curate and computationally analyse 3,653 A domains and present two high-accuracy specificity predictors, PARAS and PARASECT. A type of A domain with unusually high L-tryptophan specificity was identified through the application of PARAS, and intact protein mass spectrometry to the corresponding NRPS showed it to direct the production of tryptopeptin-related metabolites in Streptomyces species. Together, these technologies will accelerate the characterisation of novel NRPSs and their metabolic products. PARAS and PARASECT are available at https://paras.bioinformatics.nl. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/631717v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@8e48aaorg.highwire.dtl.DTLVardef@144c78dorg.highwire.dtl.DTLVardef@890c11org.highwire.dtl.DTLVardef@1776de9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Biochemical dissection of a fungal highly reducing polyketide synthase condensing region reveals basis for acyl group selection.

Fungal highly reducing polyketide synthases (hrPKSs) are remarkable multidomain enzymes that catalyse the biosynthesis of a diverse range of structurally complex compounds. During biosynthesis, the ketosynthase (KS) and acyltransferase (AT) domains of the condensing region are visited by the acyl carrier protein (ACP) domain during every cycle, catalysing chain priming and elongation reactions. Despite their significance, our comprehension of how these steps contribute to biosynthetic fidelity remains poorly understood. Herein, we report the full in vitro reconstitution of each catalytic step carried out by a hrPKS condensing region. Application of intact protein mass spectrometry shows that the AT domain operates selective acyl transfer to the ACP domain, ensuring loading of the correct starter / extender units, which is governed by a substrate recycling mechanism and kinetic parameters towards each substrate. The KS domain is shown to select for the correct starter unit for transacylation and subsequent chain elongation, yet harbours flexibility towards the extender unit supplied. This work provides the first comprehensive interrogation of a fungal hrPKS condensing region, which both affirms and expands our understanding of how these systems function, and provides a framework for future bioengineering efforts.

biochemistry↗

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↗

bioRxiv2022

Elucidating the molecular programming of a nonlinear nonribosomal peptide synthetase responsible for fungal siderophore biosynthesis.

Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) assembly lines remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC revealed its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovered several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

biochemistry↗

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↗

bioRxiv2021

Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

SUMOylation is critical for a plethora of cellular signalling pathways including the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Based on systematic proteome microarray screening combined with widely applicable carbene footprinting and high-resolution structural profiling, we define two non-conventional SUMO2-binding modules on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, interaction of SUMO2 with XRCC4 is incompatible with XRCC4 binding to at least two other NHEJ proteins - XLF and DNA ligase 4 (LIG4). These findings are consistent with SUMO2 interactions of XRCC4 acting as backup pathways at different stages of NHEJ, in the absence of these factors or their dysfunctioning. Such scenarios are not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. This work reveals insights into topology-specific SUMO recognition and its potential for modulating DSB repair by NHEJ. Moreover, it provides a rich resource on binary SUMO receptors that can be exploited for uncovering regulatory layers in a wide array of cellular processes.

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↗