Type I terpene synthases generate complex polycyclic scaffolds through carbocation cascades. However, how closely related enzymes convert a common C25 precursor to distinct sesterterpene frameworks remains unresolved. Here, we combine high-resolution crystal structures with systematic mutagenesis of four bacterial sesterterpene synthases to define the structural basis for pathway divergence. These analyses show how local active-site interactions within a conserved fold redirect carbocation trajectories and thereby control product formation. The structures reveal a preorganized binding mode that positions the substrate in a product-like conformation and directs the cyclization cascade. Comparative structural analyses further support distinct carbocation trajectories involving either centralized cation hubs or sequential rearrangement pathways with the exact pathway being enzyme-dependent. Structure-guided mutagenesis targets these features, alters product profiles, and enables the formation of new terpene scaffolds. Together, crystallographic data and mutagenesis demonstrate that closely related enzymes with the same overall fold can follow distinct carbocation trajectories and reveal how local architectural changes control the cyclization outcome.

Non-canonical terpene biosynthesis expands structural diversity beyond the classical C5n logic. Here we report the discovery of descartane, a C16 homosesquiterpene with an unprecedented scaffold produced by a bacterial type I terpene cyclase from Morganella morganii (MmTC). Unlike most terpene cyclase products, descartane is fully saturated and lacks both alkene and alcohol functions, while containing a cyclopropane ring formed in an unusual termination step. Functional characterisation demonstrated that MmTC converts α-presodorifen pyrophosphate (α-PSPP) into descartane. The cyclisation cascade was investigated by extensive isotopic labelling experiments and density functional theory (DFT) calculations, revealing a complex pathway involving fragmentation-recombination, multiple hydride shifts, and cyclopropane formation. Site-directed mutagenesis identified active site residues that control product formation and redirect the cascade towards related terpenes, including voltairene, malebranchene, de-beauvoirol, and pythagorene. These findings establish the biosynthetic origin of descartane and show how subtle changes in the active site can reprogram complex terpene cyclisation cascades.

Non-canonical methylation events generate terpene structures that evade classical biosynthetic predictions, as exemplified by the proposed C16 terpene hegelenether. Here, we show that this natural product is misassigned and revise its structure to the dihydroxylated sesquiterpenoid marxdiol. Its absolute configuration and that of its precursor prekantenol pyrophosphate were determined through terpene synthase-mediated incorporation of stereoselectively labeled probes. To explain the initiating C6 methylation, we solved the crystal structure of the methyltransferase C6-FPP-MT with SAH and FPP, revealing a compact aromatic pocket that enforces Si-face methylation and Glu165-mediated deprotonation. These insights define how the active site controls regio- and stereochemistry and provide a structural basis for identifying related methyl-modified terpenes in uncharacterized biosynthetic pathways.

Zusammenfassung Nicht‐kanonische Methylierungen erzeugen Terpene, die klassischen biosynthetischen Vorhersagen entgehen, wie die vorgeschlagene Struktur des C 16 ‐Terpens Hegelenether zeigt. Wir zeigen hier, dass diesem Naturstoff eine falsche Struktur zugeordnet worden ist und korrigieren die Struktur zum dihydroxylierten Sesquiterpenoid Marxdiol. Dessen absolute Konfiguration und die seines Vorläufers Prekantenolpyrophosphat wurden durch Terpensynthase‐vermittelten Einbau stereoselektiv markierter Sonden bestimmt. Um die initiierende C6‐Methylierung zu erklären, haben wir die Kristallstruktur der Methyltransferase C6‐FPP‐MT mit SAH und FPP gelöst. Dabei wurde eine kompakte aromatische Tasche entdeckt, die eine Si ‐seitige Methylierung und eine Glu165‐vermittelte Deprotonierung bewirkt. Diese Erkenntnisse definieren, wie das aktive Zentrum die Regio‐ und Stereochemie steuert, und liefern eine strukturelle Grundlage für die Identifizierung verwandter methylmodifizierter Terpene in bisher uncharakterisierten Biosynthesewegen.

Closthioamide (CTA) is a potent antibiotic with a unique polythioamide scaffold produced by Ruminiclostridium cellulolyticum. Unlike classical non-ribosomal peptide synthetases (NRPSs), which use modular adenylation and condensation domains, CTA biosynthesis proceeds through non-canonical standalone enzymes. Central to this process is the papain-like ligase CtaG, which catalyzes amide bond formation between two distinct peptidyl carrier proteins (PCPs): CtaH, presenting para-hydroxybenzoic acid (PHBA), and CtaE, carrying a tri-β-alanine ((βAla)3) chain. Using biochemical assays, chemical probes, crystallography, and mutational analysis, we show that CtaG operates via a ping-pong mechanism involving an enzyme-bound intermediate. A single substrate tunnel mediates directional transfer, enabling distal chain elongation that mirrors solid-phase peptide synthesis. Structure-based genome mining revealed homologous enzymes in the biosynthetic pathways of petrobactin, butirosin, and methylolanthanin. Together, our findings uncover a previously overlooked class of thiotemplated ligases and provide a mechanistic blueprint for engineering ribosome-independent peptide assembly lines.

Activity-based probes (ABPs) have become powerful tools for profiling enzymes that write and erase ubiquitin (Ub) and ubiquitin-like modifier (Ubl) signals. Extending this strategy to small ubiquitin-like modifier (SUMO)-specific proteases in defined substrate contexts remains challenging, because site-specific SUMO attachment must be combined with precise electrophile placement near the scissile isopeptide linkage. Here, we introduce a sortase-enabled chemoenzymatic platform for generating SUMO ABPs ranging from monoSUMO probes to native-like SUMO-substrate conjugates. The engineered sortase Srt2A ligates SUMO variants to glycine-bearing electrophiles, providing facile access to monoSUMO probes that trap deSUMOylases in vitro, in cellular lysates, and in living cells. To generate SUMO-substrate probes, we develop AzGVAisoK, a genetically encodable bifunctional lysine derivative containing both an azide-protected sortase handle and a vinyl amide electrophile. An engineered pyrrolysyl-tRNA synthetase/tRNA pair enables its site-specific incorporation into target proteins. Subsequent on-protein Staudinger reduction and sortase-mediated SUMOylation furnish defined SUMO-substrate ABPs under mild aqueous conditions. Applying this platform to PCNA and K11-linked diSUMO conjugates revealed distinct deSUMOylase trapping profiles governed by SUMO paralog and acceptor-substrate contexts. This work establishes a modular route to native-like SUMO probes and provides a general strategy for interrogating context-dependent enzyme recognition in Ubl signaling.

Terpene synthases usually generate carbocations by either pyrophosphate (PP) elimination (class I) or substrate protonation (class II) to initiate cyclization cascades. By contrast, recent discoveries describe methyltransferases (MTs) that act on farnesyl PP (FPP) and trigger terpene cyclization through methyl transfer. Here, we present high-resolution structures of four terpene-cyclizing FPP-MTs in complex with cofactor and substrate. The captured open and closed states reveal a dynamic MT-PP sensor that coordinates Mg2+ and PP, thereby enforcing methylation as the initial step within the catalytic chamber. Despite divergent products, these MTs share conserved active sites, with specificities determined by PP anchoring and substrate orientation. Systematic mutagenesis identified carbocation-stabilizing residues, mapped shunt pathways, and showed that subtle substitutions can redirect the cascade toward distinct terpene products. From an evolutionary perspective, the data suggest that selective pressure acted on the coordinated interplay of residues rather than on single positions, thereby stabilizing defined pathways.

Metronidazole is a front-line drug for the treatment of Helicobacter pylori infections. However, its mode of action and cellular targets are poorly defined, and higher dosing and combination therapies are required to overcome resistance. Here we performed activity-based protein profiling with tailored metronidazole probes and identified chaperonin HpGroEL and thiol peroxidase HpTpx as prominent targets, the latter being essential for H. pylori survival under oxidative stress. Alkynylated ether probes exhibited enhanced antibacterial potency compared with the parent drug in vitro, including activity against resistant strains. Biological assays, chemical proteomics and co-crystallization studies confirmed target engagement, with enhanced binding of ether derivatives to HpTpx. Refined ether analogues exhibited favourable pharmacological profiles without cytotoxicity. The in vivo activity of ether analogues using an H. pylori mouse model demonstrated full bacterial eradication at low dosing of 0.3 mg kg-1 day-1. Our findings reveal that stress induction and simultaneous inhibition of the stress response represent a mechanism of this compound class.

The proteasome inhibitors bortezomib, carfilzomib, and ixazomib all act by inhibiting multiple active sites of both constitutive proteasomes and immunoproteasomes. These clinical anticancer drugs are effective, but also display side effects, and evidence is amassing that their toxicity arises from constitutive proteasome inhibition. In this work, we describe the structure-guided discovery of a new class of pan-immunoproteasome-selective inhibitors. We identified the peptide epoxyketone BocPip-Ser (8), which targets all three human immunoproteasome active sites potently and with excellent selectivity over constitutive proteasome active sites (IC50 values for i-subunits ≤ 0.92 μM; IC50 ratio β1c/β1i: 13, β2c/β2i: 14, β5c/β5i: 18; Table 1 and Figure 3). We propose compound 8 (BocPip-Ser), which is of a similar size and general properties as carfilzomib, as a lead compound for the development of improved drugs targeting hematological cancers, and possibly also autoimmune diseases, driven by immunoproteasome but not constitutive proteasome activities.

ZUSAMMENFASSUNG Die nicht‐kanonische Terpenbiosynthese erweitert die strukturelle Vielfalt über die klassische C 5n ‐Logik hinaus. Wir berichten hier über die Entdeckung von Descartan, einem C 16 ‐Homosesquiterpen mit einem neuartigen Grundgerüst, das von einer bakteriellen Terpencyclase vom Typ I aus Morganella morganii (MmTC) produziert wird. Im Gegensatz zu den meisten Terpencyclaseprodukten ist Descartan vollständig gesättigt und weist weder Alken‐ noch Alkoholfunktionen auf, enthält aber einen Cyclopropanring, der in einem ungewöhnlichen Abbruchschritt gebildet wird. Funktionelle Charakterisierungen zeigten, dass MmTC α‐Presodorifenpyrophosphat (α‐PSPP) in Descartan umwandelt. Die Cyclisierungskaskade wurde mittels umfangreicher Isotopenmarkierungsexperimente und Dichtefunktionaltheorie‐Berechnungen (DFT) untersucht. Dabei wurde ein komplexer Reaktionsweg aufgezeigt, der Fragmentierung und Rekombination, mehrfache Hydridverschiebungen und die Bildung eines Cyclopropanrings umfasst. Durch gezielte Mutagenese wurden Aminosäurereste im aktiven Zentrum identifiziert, die die Produktbildung steuern und die Reaktionskaskade hin zu verwandten Terpenen wie Voltairen, Malebranchen, De‐Beauvoirol und Pythagoren lenken. Diese Ergebnisse belegen den biosynthetischen Ursprung von Descartan und zeigen, wie subtile Veränderungen im aktiven Zentrum komplexe Terpencyclisierungs‐Kaskaden umprogrammieren können.

Incorporating metal cofactors into computationally designed protein scaffolds provides a versatile route to novel protein functions, including the potential for new-to-nature enzyme catalysis. However, a major challenge in protein design is to understand how the scaffold architecture influences conformational dynamics. Here, we characterized structure and dynamics of a modular de novo scaffold with flexible inter-domain linkers. Three rationally engineered variants with different metal specificity were studied by combining X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations. The lanthanide-binding variant was initially trapped in an inactive conformational state, which impaired efficient metal coordination and cerium-dependent photocatalytic activity. Stabilization of the active conformation by AI-guided sequence optimization using ProteinMPNN led to accelerated lanthanide binding and a 10-fold increase in kcat/Km for a photoenzymatic model reaction. Our results suggest that modular scaffold architectures provide an attractive starting point for de novo metalloenzyme engineering and that ProteinMPNN-based sequence redesign can stabilize desired conformational states.

Tracking small-molecule distribution in heterogeneous cell samples at single-cell resolution remains a major analytical challenge. Here, we present a tellurophene-functionalized analogue of the proteasome inhibitor Carfilzomib (TeCar) whose distribution can be followed by mass cytometric (MC) quantification while preserving target engagement and cytotoxicity. Structural and biochemical analyses confirm that TeCar binds the proteasome in a mode comparable to the clinically approved parent compound. Using MC, we demonstrate selective TeCar accumulation in malignant over immune cells within mixed populations, with cancer cells exhibiting 15 to 30-fold higher uptake. Tellurium signal correlates with proteasomal activity, and differential labeling among immune subsets reveals functional heterogeneity not captured by transcriptomics alone. These findings establish tellurophene tagging as a minimally perturbing and broadly applicable strategy for functional distribution studies at single-cell resolution.

Human sirtuin 2 (SIRT2) is an NAD+ dependant enzyme that has been linked to the pathogenesis of various diseases, making it a promising target for pharmaceutical intervention. This study presents a systematic investigation on the inhibitory effects of SIRT2 inhibitors functionalized with diverse electrophilic functional groups. Guided by initial docking studies, we designed and synthesised 14 derivatives of two published potent lead structures 24a and SirReal2. The most potent and subtype selective SIRT2 inhibitor 29 (RW-78) exhibits an IC50 of 26 nM, which outperforms its lead structure 24a (IC50 = 79 nM) by a factor of 3. The increased potency of 29 is explained by halogen-π interactions with SIRT2 residues as visualized by X-ray crystallography. Furthermore, 29 interferes with NAD+ binding, highlighting co-factor displacement as a valid strategy to inhibit SIRT2. Additionally, we showed cellular target engagement via NanoBRET assays in HEK293T cells (EC50 = 15 nM). Altogether our findings provide a deeper insight into the structure-activity relationships of these SirReal-type inhibitors and offer new avenues for optimisation of SIRT2 inhibitors.

Epoxomicin is a highly potent natural proteasome inhibitor and the structural scaffold for the anticancer drug carfilzomib. The biosynthesis of its α′,β′-epoxyketone warhead involves the flavoenzyme EpxF, but a molecular understanding of the key catalytic reaction cascade remained elusive. Here, we disclose detailed mechanistic insights by characterizing all intermediates in the sequential steps of decarboxylation, desaturation, and epoxidation with synthetic flavins and the flavin-dependent oxidoreductase EpxF. A high-resolution crystal structure of EpxF revealed the architecture of the active site and enabled the identification of key catalytic residues. Exploratory docking based on this structure served as a qualitative tool to guide mutagenesis and rationalize substrate recognition. NMR studies with a 13 C-labeled epoxomicin precursor and structure-based EpxF variants further supported the proposed mechanism. Our integrated approach revealed similarities between synthetic and natural flavin catalysts and offers avenues for developing sustainable biomimetic reactions.

Photorhabdus strains, Gram-negative bacteria pathogenic to insect larvae, produce two signature compounds: the multifunctional isopropylstilbene (IPS), known for its antibiotic, insecticidal, and immunosuppressive activities, and orange-to-red pigmented anthraquinones (AQs), which attenuate oxidative stress. Here, we demonstrate an inverse correlation between the production of AQs and cinnamic acid (CA), the primary precursor for IPS formation in the model strain P. laumondii TTO1. Metabolic and proteomic analyses following CA treatment show that CA inhibits AntI, a key enzyme in the final step of AQ-256 biosynthesis. The crystal structure of AntI in complex with CA reveals that cinnamic acid functions as a competitive inhibitor by inducing specific structural rearrangements in the lyase, resulting in noncovalent, reversible inhibition. These findings provide atomic insights into the intricate regulatory control of pigment biosynthesis and the production of bioactive compounds.

Terpene synthases orchestrate complex cyclization cascades that transform simple polyisoprenoid precursors into structurally diverse natural products, often with exquisite stereochemical control. Here we combine high-resolution X-ray crystallography, site-directed mutagenesis, and QM/MM calculations to dissect the catalytic mechanisms of two bacterial sesquiterpene synthases for T-muurolol (TmS) and 1-epi-cubenol (NcECS). The structures reveal a dynamic transition between open and closed states, controlled by a trinuclear magnesium cluster that mediates substrate binding, carbocation formation, and intramolecular pyrophosphate transfer to generate (R)-nerolidyl pyrophosphate, the precursor to Z-configured products. Using synthetic dihydro-surrogates, we identify a counterclockwise substrate orientation, not previously observed in terpene synthases, and visualize a series of trapped hydrocarbons that resemble several of the proposed cationic intermediates along the cyclization cascade. Complementary quantum chemical calculations support their observed geometries and indicate that the active site can transiently accommodate these intermediate analogs, offering a structural basis for understanding how sesquiterpene synthases guide complex carbocationic pathways.

Xenorhabdus strains, Gram-negative bacteria pathogenic to insects and symbionts to nematodes of the genus Steinernema are prolific producers of various natural products. Here we describe the xisABCDE biosynthesis gene cluster from Xenorhabdus hominickii responsible for the production of xildivalines. These non-ribosomal peptide and polyketide hybrids act as peptide deformylase inhibitor (PDI) and occur also in other Gammaproteobacteria, especially Vibrio . Their structure and biosynthesis were fully elucidated despite their instability, highlighting a rare trans -methylation of their N-terminus. Subsequently, the structure of the responsible methyltransferase XisE and the peptide deformylase XisD, serving as resistance mechanism, were elucidated by X-ray crystallography, allowing insights into the function and the mode of action of this novel class of PDIs. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 835108 Fondation pour la Recherche Médicale, EQU202103012569 and FDM202106013531 Deutsches Elektronen-Synchrotron DESY, https://ror.org/01js2sh04, MX-970

Vanadium-dependent haloperoxidases (VHPOs) are biotechnologically valuable and operationally versatile biocatalysts. VHPOs share remarkable active-site structural similarities yet display variable reactivity and selectivity. The factors dictating substrate specificity and, thus, a general understanding of VHPO reaction control still need to be discovered. This work's strategic single-point mutation in the cyanobacterial bromoperoxidase AmVHPO facilitates a selectivity switch to allow aryl chlorination. This mutation induces loop formation that interacts with the neighboring protein monomer, creating a tunnel to the active sites. Structural analysis of the substrate-R425S-mutant complex reveals a substrate-binding site at the interface of two adjacent units. There, residues Glu139 and Phe401 interact with arenes, extending the substrate residence time close to the vanadate cofactor and stabilizing intermediates. Our findings validate the long-debated existence of direct substrate binding and provide a detailed VHPO mechanistic understanding. This work will pave the way for a broader application of VHPOs in diverse chemical processes.

Azetidine-2-carboxylic acid (AZE) is a long-known plant metabolite. Recently, AZE synthases have been identified in bacterial natural product pathways involving non-ribosomal peptide synthetases. AZE synthases catalyse the intramolecular 4-exo-tet cyclisation of S-adenosylmethionine (SAM), yielding a highly strained heterocycle. Here, we combine structural and biochemical analyses with quantum mechanical calculations and mutagenesis studies to reveal catalytic insights into AZE synthases. The cyclisation of SAM is facilitated by an exceptional substrate conformation and supported by desolvation effects as well as cation-π interactions. In addition, we uncover related SAM lyases in diverse bacterial phyla, suggesting a wider prevalence of AZE-containing metabolites than previously expected. To explore the potential of AZE as a proline mimic in combinatorial biosynthesis, we introduce an AZE synthase into the pyrrolizixenamide pathway and thereby engineer analogues of azabicyclenes. Taken together, our findings provide a molecular framework to understand and exploit SAM-dependent cyclisation reactions. Azetidine-2-carboxylic acid synthases catalyse the formation of the proline analogue azetidine-2-carboxylic acid (AZE) in bacteria. In this work, the authors combine structural and biochemical analyses with quantum mechanical calculations and mutagenesis studies to obtain catalytic insights into AZE synthases.

Single point mutations in proteasome subunits can cause severe autoinflammatory syndromes. By still largely unknown mechanisms, some of these disease-associated mutations impair normal proteasome function and induce the production of pro-inflammatory cytokines, thereby leading to systemic inflammations. In order to obtain more insights on why and how the mutations T3M and G128V in the immunoproteasome subunit β5i trigger such deleterious effects, we created the respective yeast mutants and characterized their phenotypes with special emphasis on proteasome structure and activity. X-ray crystallographic data revealed that the mutation T3M influences structure and flexibility of the proteasomal substrate-binding channel with moderate impairment of proteasome biogenesis, whereas the amino acid substitution G128V causes larger structural rearrangements that severely disturb particle assembly and maturation. The obtained results provide a deeper understanding of how single point mutations can affect proteasome subunit structure as well as particle biogenesis and ultimately cause chronic inflammatory diseases.