The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

Oxygen‐directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O‐methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized the enzymatic properties and substrate scope of two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis and determined their crystal structures. Both OMTs methylated a wide range of catechol‐like substrates, including flavonoids, coumarins, hydroxybenzoic acids, and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1‐methyl ether, and 2‐methoxyestradiol. Interestingly, certain non‐catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering.

Stagnation in the development of new antibiotics emphasizes the need for the discovery of drugs with novel modes of action that can tackle antibiotic resistance. Contrary to humans, most bacteria use the methylerythritol phosphate (MEP) pathway to synthesize crucial isoprenoid precursors. 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first and rate-limiting step of the pathway, making it an attractive target. Alkylacetylphosphonates (alkylAPs) are a class of pyruvate mimicking DXPS inhibitors that react with thiamin diphosphate (ThDP) to form a stable phosphonolactyl (PLThDP) adduct. Here, we present the first M. tuberculosis DXPS crystal structure in complex with an inhibitor (butylacetylphosphonate (BAP)) using a construct with improved crystallization properties. The 1.6 Å structure shows that the BAP adduct interacts with catalytically important His40 and several other conserved residues of the active site. In addition, a glycerol molecule, present in the D-glyceraldehyde 3-phosphate (D-GAP) binding site and within 4 Å of the BAP adduct, indicates that there is space to extend and develop more potent alkylAPs. The structure reveals the BAP binding mode and provides insights for enhancing the activity of alkylAPs against M. tuberculosis, aiding in the development of novel antibiotics.

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis in terms of their enzymatic properties and substrate scope and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering. Table of contents: Two promiscuous O-methyltransferases from bacteria were found to methylate a panel of catechol substrates towards high-value medicinal compounds. Surprisingly, the non-catechol substrates 5-hydroxyflavonoids and o -hydroxybenzoic acids/aldehydes were also methylated at low conversion rates. The crystal structures reveal potential target sites for enzyme engineering for biocatalytic applications.

Among the promising microbial platforms to produce value-added products from CO ₂ , chemolithoautotrophic bacteria such as Cupriavidus necator H16 ( C. necator ) stand out due to their ability to fix CO ₂ via the Calvin-Benson-Bassham (CBB) cycle. Although C. necator has been extensively studied, many enzymes and associated biosynthetic pathways have yet to be identified and fully understood, which is necessary to engineer this microorganism for efficient biotechnological production. One particularly interesting target is the biosynthetic enzymes involved in L-methionine synthesis as this amino acid is currently the only one still produced exclusively from petroleum. In this study, we identified and characterized the enzymatic properties of Cn MetXS, a homoserine O -succinyltransferase and the key enzyme in the C. necator biosynthetic pathway of L-methionine and determined its crystal structure. Cn MetXS catalyzes the transsuccinylation of L-homoserine to O -succinyl-L-homoserine but exhibits no transacetylase activity. This study verifies Cn MetXS as an O -succinyltransferase and paves the way for metabolic engineering strategies for the microbial production of L-methionine in C. necator .

Multicomponent Rieske oxygenases catalyze diverse oxidative transformations but require precisely matched redox partners to sustain efficient electron transfer, severely limiting their modularity and biocatalytic application. Yet, the molecular logic underlying this specificity remains poorly defined. Here we decode the molecular principles governing redox partner specificity in representative three-component Rieske oxygenase systems. Through systematic mutagenesis analysis and cross-component reconstitution assays, we identify a single ferredoxin residue that acts as a class-defining determinant of oxygenase recognition. Guided by this insight, we reprogram electron transfer between non-cognate components by complementary engineering of the oxygenase interface, creating an unnatural redox chain with substantially enhanced catalytic turnover compared to the native system. Spectroscopic, binding and computational analyses reveal that productive electron transfer arises from optimized electrostatic complementarity and redox potential alignment rather than maximal binding affinity. Extending this strategy to another oxygenase system demonstrates its generality. Together, these results establish transferable design rules for rationally engineering electron transfer pathways in multicomponent oxygenases, enabling their predictable adaptation as customizable biocatalysts.