Biology subjects

Fage, C. D.

Publications and source records attributed to Fage, C. D..

4 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

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

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↗

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↗