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Dithiolopyrrolone (DTP) natural products are produced by several different bacteria and have potent antibacterial, antifungal and anticancer activities. While the amide of their DTP core can be methylated to fine‐tune bioactivity, the enzyme responsible for the amide N‐methylation has remained elusive in most taxa. Here, we identified the amide methyltransferase XrdM that is responsible for xenorhabdin (XRD) methylation in Xenorhabdus doucetiae but encoded outside of the XRD gene cluster. XrdM turned out to be isofunctional with the recently reported methyltransferase DtpM, that is involved in the biosynthesis of the DTP thiolutin, although its X‐ray structure is unrelated to that of DtpM. To investigate the structural basis for ligand binding in both enzymes, we used X‐ray crystallography, modeling, site‐directed mutagenesis, and kinetic activity assays. Our study expands the limited knowledge of post‐non‐ribosomal peptide synthetase (NRPS) amide methylation in DTP biosynthesis and reveals an example of convergent evolution of two structurally completely different enzymes for the same reaction in different organisms. [ABSTRACT FROM AUTHOR]

Fungi utilize high‐affinity chelators termed siderophores with chemically diverse structures to scavenge the essential nutrient iron from their surroundings. Since they are among the strongest known Fe3+ binding agents, intracellular release of the heavy metal atom is facilitated by the activity of specific hydrolases. In this work, we report the characterization and X‐ray crystal structures of four siderophore esterases: AfEstB and AfSidJ from Aspergillus fumigatus, as well as AnEstB and AnEstA from Aspergillus nidulans. Even though they all display the conserved α/β‐hydrolase fold, we found significant structural and enzymatic discrepancies in their adaption to both related and chemically diverse substrates. A structure of AfEstB in complex with its substrate triacetylfusarinine C gives insight into the active enzyme and shows tetrahedral coordination between the catalytic serine and the scissile ester bond. Breaking the cage: Fungi use siderophores to scavenge Fe3+ from the environment. Intracellular iron release occurs through hydrolysis of the siderophore backbone by esterases. Structural and biochemical analyses of two homologous EstB esterases, as well as EstA and SidJ, reveal how a common fold adapts to diverse substrates. A structure of an active enzyme in complex with its siderophore substrate reveals a tetrahedral intermediate at the catalytic serine. [ABSTRACT FROM AUTHOR]

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Mammaliangenomes encode seven catalytic proteasome subunits, namely,β1c, β2c, β5c (assembled into constitutive 20S proteasomecore particles), β1i, β2i, β5i (incorporated intoimmunoproteasomes), and the thymoproteasome-specific subunit β5t.Extensive research in the past decades has yielded numerous potentproteasome inhibitors including compounds currently used in the clinicto treat multiple myeloma and mantle cell lymphoma. Proteasome inhibitorsthat selectively target combinations of β1c/β1i, β2c/β2i,or β5c/β5i are available, yet ligands truly selectivefor a single proteasome activity are scarce. In this work we reportthe development of cell-permeable β1i and β5i selectiveinhibitors that outperform existing leads in terms of selectivityand/or potency. These compounds are the result of a rational designstrategy using known inhibitors as starting points and introducingstructural features according to the X-ray structures of the murineconstitutive and immunoproteasome 20S core particles. [ABSTRACT FROM AUTHOR]

Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Azoles are first-line therapeutics for human and plant fungal infections, but their broad use has promoted the development of resistances. Recently, a pan-azole-resistant clinical Aspergillus fumigatus isolate was identified to carry the mutation P88L in subunit HapE of the CCAAT-binding complex (CBC), a conserved eukaryotic transcription factor. Here, we define the mechanistic basis for resistance in this isolate by showing that the HapE P88L mutation interferes with the CBC's ability to bend and sense CCAAT motifs. This failure leads to transcriptional derepression of the cyp51A gene, which encodes the target of azoles, the 14-α sterol demethylase Cyp51A, and ultimately causes drug resistance. In addition, we demonstrate that the CBC-associated transcriptional regulator HapX assists cyp51A repression in low-iron environments and that this iron-dependent effect is lost in the HapE P88L mutant. Altogether, these results indicate that the mutation HapE P88L confers increased resistance to azoles compared with wt A. fumigatus , particularly in low-iron clinical niches such as the lung., (© 2020 Hortschansky et al.)

Fungi utilize high-affinity chelators termed siderophores with chemically diverse structures to scavenge the essential nutrient iron from their surroundings. Since they are among the strongest known Fe 3+ binding agents, intracellular release of the heavy metal atom is facilitated by the activity of specific hydrolases. In this work, we report the characterization and X-ray crystal structures of four siderophore esterases: AfEstB and AfSidJ from Aspergillus fumigatus, as well as AnEstB and AnEstA from Aspergillus nidulans. Even though they all display the conserved α/β-hydrolase fold, we found significant structural and enzymatic discrepancies in their adaption to both related and chemically diverse substrates. A structure of AfEstB in complex with its substrate triacetylfusarinine C gives insight into the active enzyme and shows tetrahedral coordination between the catalytic serine and the scissile ester bond., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Clinically applied proteasome inhibitors induce cell death by concomitant blockage of constitutive and immunoproteasomes. In contrast, selective immunoproteasome inhibition is less cytotoxic and has the potential to modulate chronic inflammation and autoimmune diseases. In this study, we rationally designed decarboxylated peptides that covalently target a non-catalytic cysteine of the immunoproteasome subunit β5i with α-chloroacetamide-containing sidechains. The enhanced isoform specificity decreased cytotoxic effects and the compound suppressed the production of inflammatory cytokines. Structure-based optimization led to over 150-fold selectivity for subunit β5i over β5c. This new compound class provides a promising starting point for the development of selective immunoproteasome inhibitors as potential anti-inflammatory agents., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Broad-spectrum proteasome inhibitors are applied as anticancer drugs, whereas selective blockage of the immunoproteasome represents a promising therapeutic rationale for autoimmune diseases. We here aimed at identifying minimal structural elements that confer β5c or β5i selectivity on proteasome inhibitors. Based on the natural product belactosin C, we synthesized two β-lactones featuring a dimethoxybenzyl moiety and either a methylpropyl (pseudo-isoleucin) or an isopropyl (pseudo-valine) P1 side chain. Although the two compounds differ only by one methyl group, the isoleucine analogue is six times more potent for β5i (IC50=14 nM) than the valine counterpart. Cell culture experiments demonstrate the cell-permeability of the compounds and X-ray crystallography data highlight them as minimal fragments that occupy primed and non-primed pockets of the active sites of the proteasome. Together, these results qualify β-lactones as a promising lead-structure motif for potent nonpeptidic proteasome inhibitors with diverse pharmaceutical applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nature provides a rich source of compounds with diverse chemical structures and biological activities, among them, sulfur-containing metabolites from bacteria and fungi. Some of these compounds bear a disulfide moiety that is indispensable for their bioactivity. Specialized oxidoreductases such as GliT, HlmI, and DepH catalyze the formation of this disulfide bridge in the virulence factor gliotoxin, the antibiotic holomycin, and the anticancer drug romidepsin, respectively. We have examined all three enzymes by X-ray crystallography and activity assays. Despite their differently sized substrate binding clefts and hence, their diverse substrate preferences, a unifying reaction mechanism is proposed based on the obtained crystal structures and further supported by mutagenesis experiments., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)