Biology subjects

Alkhalaf, L. M.

Publications and source records attributed to Alkhalaf, L. M..

9 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↗

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↗

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

Nitrogen deletion in the biosynthesis of HDAC-targeting anticancer drugs

Small molecules play indispensable roles in living systems as hormones, membrane bilayer constituents, enzyme cofactors, metal chelators, and defensive chemicals. The exceptional structural diversity of such molecules underpins their wide-ranging biological functions. Precise functional group insertion into various molecular scaffold classes is a hallmark of small molecule biosynthesis and is frequently important for biological activity. Functional group deletion is also important, but mechanisms are less well understood. Here, we report deletion of a cysteine-derived nitrogen atom during assembly of the conserved pharmacophore in the anticancer drug romidepsin, and related depsipeptide HDAC inhibitors. A shunt metabolite hydroxylated at the cysteine--carbon-derived position is a thousand-fold less active, indicating nitrogen deletion is important for potent HDAC inhibition. In vitro reconstitution and dissection of the complete nonribosomal peptide synthetase-polyketide synthase-mediated pathway for pharmacophore assembly reveal that cryptic S-octanoylation is catalysed by an atypical heterocyclisation domain, while multifunctional dehydratase and ketoreductase domains and trans-acting phosphotransferase and flavin-dependent oxidoreductase enzymes catalyse successive transformations in nitrogen deletion. Our findings significantly advance the understanding of heteroatom deletion mechanisms in small molecule biosynthesis and highlight the key role this can play in enhancing bioactivity. One-pot biocatalytic synthesis of the pharmacophore provides foundations for chemoenzymatic approaches to next-generation HDAC inhibitors.

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↗

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↗

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↗