Angewandte Chemie International Edition

While lipidation is a widely observed strategy to promote membrane permeation, whether the factors governing lipid-driven accumulation are shared across the divergent membranes of mammalian and Gram-negative cells remains unresolved. Here, we apply the Chloroalkane Azide-based Membrane Penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. coli accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. Through targeted endogenous and exogenous modifications, we further resolve how charge, scaffold composition, and individual envelope barriers shape these patterns. Together, these results establish that lipidation is a context-dependent permeation principle that fundamentally diverges between mammalian and diderm envelopes. By showing that hydrophobic modifications routinely hinder Gram-negative cytosolic entry, this work explains the scarcity of lipidated Gram-negative antimicrobials, exposes the limits of lipophilicity-driven optimization, and redefines the physicochemical boundaries for penetrating the diderm envelope.

Pyrrolizidine alkaloids (PA) are well-known and widespread natural products from plants, which have also been identified in several different bacteria. In the latter case, the core structure is constructed by a non-ribosomal peptide synthetase (NRPS), which then undergoes oxidative ring contraction catalyzed by a Baeyer-Villiger monooxygenase. By deploying various NRPS engineering strategies, we have successfully generated five novel peptides carrying the unusual PA moiety at their C-terminus. Nonetheless, efforts to obtain a larger library of PAs were unsuccessful. Combined computational modelling and docking experiments suggest that this failure stems from the strict specificity of the thioesterase (TE) domain at the end of the NRPS, which discriminates against peptides carrying more than two amino acids. Our work thus suggests protein design strategies by which this intrinsic limitation to NRPS engineering may be overcome in future.

Terpenoids are ubiquitous, structurally diverse natural products found across the three domains of life. One of the most commonly observed and well-studied terpenoids is the volatile, petrichoric compound geosmin, responsible for the earthy smell of soil to which humans are receptive down to a few parts per trillion. Bioinformatic analysis of bacterial geosmin synthase (GeoA)-encoding gene clusters reveals that many are colocalized with genes encoding Family 2B encapsulin shell proteins. Here, we focus on the encapsulin-encoding geosmin gene cluster of the model myxobacterium Myxococcus xanthus and show that the two shell proteins form a two-component Family 2B encapsulin with GeoA as the internalized cargo. Structural analysis highlights that the shell contains characteristic, external cyclic adenosine monophosphate (cAMP) binding-fold domains (CBDs), and that mixed shells can adopt a previously unobserved closed two-fold pore conformation, potentially important in cargo function modulation and regulation. We show that GeoA cargo loading is mediated by repeating, short cargo loading peptides (CLPs), and that GeoA encapsulation provides benefits for enzyme activity and stability. This work expands the short list of Family 2B encapsulins known to be involved in specialized metabolite biosynthesis, and provides insights into a putative mode of cargo protein regulation via pore state modulation.

Many biological proteins function by changing shape upon small-molecule binding. Here, we present a general strategy for designing de novo proteins that undergo small-molecule-induced conformational change. Our approach converts a preorganized small-molecule binding protein into a ligand-responsive shape-changer by adding a mobile lid domain that closes behind the ligand upon binding. Using this strategy, we converted an exatecan-binding protein into a drug-induced conformational switch. The lidded proteins showed considerably stronger binding affinity in the sub-nanomolar regime, 100-fold greater specificity to exatecan over a similar molecule, and ligand residence times up to several months, with tunable binding kinetics. We turned one design into a genetically encodable fluorescent biosensor of the drug, enabling potential clinical applications. Our results open the door to programming complex molecular function using vast chemical space.

The ability to image specific lipid subtypes within cells can have a transformative impact on the study of lipid dynamics and trafficking mechanisms. Herein, we describe a technology for imaging phosphatidylethanolamine (PE) lipids in live mammalian cells that involved screening a library of ethanolamine derivatives to identify an azido compound that efficiently metabolically labels PE. Crucially, this probe evades the cellular methylation machinery specifically labelling PE without forming labelled methylated PE and phosphatidylcholine (PC) lipids. The administration of cyclooctyne dyes to cells metabolically labelled with this probe rendered azido PE lipids fluorescent via strain-promoted click chemistry, enabling imaging. We employed this technology to image PE in various cellular organelles, visualize PE externalization during apoptosis, and discover that the VPS13A protein transports PE from the endoplasmic reticulum to the mitochondria. This technology will facilitate addressing fundamental questions in PE biology and studying dysregulation of PE dynamics and trafficking in disease states.

Taxol (paclitaxel) is a frontline anticancer drug widely applied for the treatment of breast, ovarian and lung cancers. Currently, its supply mainly relies on the semi-synthesis using baccatin III from Taxus plants. Heterologous biosynthesis of baccatin III in microorganisms offers a promising solution to alleviate global Taxol supply shortage, but remains challenging due to pathway complexity. Here, we report a novel taxusin-mediated biosynthetic pathway for baccatin III production via the identification of C13 deacetylase, elucidation of the exact sequence underlying C1 hydroxylation, and stepwise enzymatic functional validation. Through protein engineering of the promiscuous C1 and C5 hydroxylases, coupled with the distribution of pathway modules in Saccharomyces cerevisiae and Escherichia coli, we achieved the de novo biosynthesis of baccatin III. Collectively, our findings remodel the current biosynthetic framework governing the formation of Taxol precursors and highlight the great potential of microbial cell factories for the production of complex plant-derived therapeutic compounds. Highlights* Discovery of C13 deacetylase reveals a novel biosynthetic route to baccatin III via taxusin * Stepwise verification of the complete biosynthetic route to baccatin III through taxusin and baccatin VI * Single-site mutation reversed the product selectivity of T1OH and converted T5OH into a specific taxoid C5 hydroxylase * Complete biosynthesis of baccatin III in engineered microbes

The de novo design of proteins that bind chemically complex small molecules has broad chemical and biological implications, but strategies typically rely on a small set of protein scaffolds and require extensive experimental screening. Here, we computationally designed proteins around a minimal aromatic {pi}-stacking motif to bind the anthracycline anticancer drug doxorubicin. Experimental characterization of twelve proteins revealed a {micro}M doxorubicin binder; two additional design cycles improved scaffold stability and binding affinity to yield an 85-residue protein that binds doxorubicin with a dissociation constant of 85 nM. An X-ray crystal structure of the protein-drug complex confirmed the accuracy of the designed {pi}-{pi} stacking interactions. The designed protein could act to protect cultured cells from doxorubicin-induced cytotoxicity. Unlike previous ligand-binding protein designs based on repeat proteins or naturally occurring folds, the designed protein adopts a previously unobserved 5-helix globular fold, indicating that a broader space of folded, functional proteins exists even for compact tertiary structures smaller than 100 residues. These results demonstrate that motif-guided generative protein design can discover compact de novo protein folds capable of high-affinity recognition of chemically complex small molecules.

Glycine is a vital extracellular signal in bacteria, plants, and the brain. Although RNA-based sensors detect glycine in cells, their extracellular application in native biological environments is limited by enzymatic degradation from nucleases. Mirror-image RNA is nuclease-resistant and preserves the tertiary structure required for RNA function, but synthesizing long L-RNAs such as the 170-nt glycine sensor (glyS) remains challenging. Here, we applied cross-chiral ligation with natural D-RNA ribozymes to assemble a mirror-image L-RNA glycine sensor (L-glyS). Optimization of the ligation conditions enabled up to 68% conversion to the full-length sensor. L-glyS displayed nuclease resistance and maintained glycine-dependent fluorescence in serum, where the original D-glyS lost function. These results establish cross-chiral ligation as a strategy for constructing long, functional L-RNAs and broaden the possible applications of RNA-based sensors to extracellular detection of small molecules.

Skeletal rearrangement drives the immense structural complexity of terpene, yet predicting it remains a formidable challenge due to sequence-function decoupling in terpene synthases. Here, we established TRACER (terpene rearrangement annotation via co-attentive enzyme-product representation), a multimodal framework mapping the latent associations between sequence-derived enzyme representations and product chemotypes. Retrospective validation proved TRACERs exceptional precision in predicting compound classes and discriminating skeletal rearrangement (SR) from non-skeletal rearrangement (NSR) pathways. TRACER-guided genome mining characterized two bifunctional synthases, FsPS and AcPS, uncovering four unprecedented carbon skeletons. Density functional theory calculations deciphered these cyclization cascades, pinpointing a critical 5/6/11 tricyclic intermediate as the key branching node for scaffold diversification. Mutagenesis and molecular dynamics simulations suggested that E305 in FsPS enables rearrangement by maintaining active-site water exclusion, whereas its alanine mutation causes premature carbocation quenching. Collectively, this work establishes a predictive paradigm for the rational discovery and mechanistic elucidation of complex terpene architectures.

We present a robust and versatile in vitro transcription (IVT) assay based on an optimized Broccoli RNA aptamer sequence. When paired with the fluorophore DFHBI-1T, this system enables real-time monitoring of multi-round transcription over several hours. To facilitate streamlined promoter analysis, we developed the pIVT3 plasmid backbone. The system was validated using both the single-subunit T7 RNA polymerase and the multi-subunit Escherichia coli RNA polymerase; notably, the activity of the E. coli enzyme remained strictly dependent on the presence of a {sigma} factor and a cognate promoter. To optimize the signal-to-noise ratio, we incorporated two rrnBT1 terminators upstream of the promoter of interest. This modification effectively eliminated background transcription for weak promoters (PlivJ) and prevented interference from read-through transcription in strong synthetic promoters (Ptrc*). Furthermore, we demonstrated the assays utility for drug discovery by characterizing the time- and dose-dependent inhibitory kinetics of rifampicin. Collectively, these results establish the Broccoli-based IVT system as a highly adaptable platform for quantifying promoter strength and screening small-molecule inhibitors of bacterial transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/744185v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1e0c991org.highwire.dtl.DTLVardef@d154aeorg.highwire.dtl.DTLVardef@10e95fcorg.highwire.dtl.DTLVardef@98ea80_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nature produces hundreds of carotenoids, yet only a handful of the apocarotenoids derived from them are accessible through microbial production. The best-known example is retinal, the chromophore of rhodopsins and a precursor of pharmaceutical retinoids, which is generated by the central cleavage of {beta}-carotene. Whether the same cleavage chemistry can be extended to other carotenoids, yielding retinal analogues that differ in their ring structures, and potentially in their biological activities, has remained largely untested. In this study, we demonstrate a pathway engineering approach in E. coli for the biosynthesis of diverse retinal analogues by leveraging substrate promiscuity of Blh, a bacterial carotenoid cleavage enzyme originally identified in microbial rhodopsin gene clusters. While initial co-expression of Blh with carotenoid pathway genes often resulted in the production of retinal (by cleavage of {beta}-carotene intermediate), we found that by optimizing the expression level of Blh, carotenoids such as astaxanthin or canthaxanthin were cleaved efficiently. Structure-guided engineering of Blh, informed by its predicted substrate-binding cavity, further improved the cleavage of zeaxanthin. This expanded catalytic activity suggests that Blh can serve as a versatile biocatalyst for the production of diverse retinal analogues, potentially yielding compounds with a range of biological activities. Furthermore, our findings raise the possibility of diverse biological roles for these enzymes in their native biological contexts. ImportanceThis study demonstrated the successful biosynthesis of a diverse array of retinal analogues in engineered Escherichia coli through the heterologous expression of Blh, a {beta}-carotene cleavage dioxygenase, together with several carotenoid pathways. Careful design of the Blh expression construct enabled modulation of retinoid proportions in the engineered pathway. This work uncovers previously unrecognized substrate promiscuity of Blh, revealing its capacity to accept carotenoids beyond {beta}-carotene as substrates. For the first time, the predicted structure of Blh revealed the enzymes substrate cavity. Rational engineering by amino acid substitution designed to expand the cavity enabled the improved cleavage of hydroxylated carotenoids. These findings open new avenues for both fundamental research and biotechnological applications and have the potential to impact the microbial production of valuable retinoids.

Bacterial azabicyclocarbamates and related pyrrolizidine alkaloids play important roles in microbial interactions, and are scaffolds of therapeutic potential. Their biosynthesis involves a bimodular non-ribosomal peptide synthetase (NRPS), as well as a Baeyer-Villiger monooxygenase and tailoring enzymes, the latter contributing to the structural diversification of these compounds. Azetidomonamide A, a core metabolite produced by the major human opportunistic pathogen Pseudomonas aeruginosa, is a rare 4,7-bicyclocarbamate involved in modulating bacterial virulence that belongs to a unique family of natural products targeting ClpP proteases. In this study, we elucidated the full set of reactions leading to the 7-membered cyclocarbamate warhead, and reconstituted in vitro the biosynthesis of azetidomonamide A. Notably, this approach allowed for detailed characterization of a condensation (C) domain-catalyzed online dehydration via chemical capture of NRPS-tethered intermediates. Furthermore, we identified the dehydratase AzeD as the founding member of a distinct group of standalone proteins of the C domain family. Via combined structural, docking and biochemical analyses, we provided evidence that AzeDs catalytic mechanism is distinct from that of dehydrating C domains, further expanding the known chemistry of these key biosynthetic enzymes.

Switchable {beta}-barrel-type fluorescent proteins are essential genetically encoded probes for super-resolution imaging. The space required for chromophore cis-trans isomerisation can also provide an opportunity to introduce bulkier chemistry at the 3-position of the phenolic ring. Here, we report, to our knowledge, the first successful genetic encoding of 3-cyano-L-tyrosine (3CNY) into a protein. Using genetic code expansion, the cyano-containing amino acid was incorporated directly into the chromophore of mKate, a pH-dependent switchable red fluorescent protein. In mKate, the chromophore adopts a fluorescent phenolate cis state at physiological pH, transitioning to a phenolic trans state under acidic conditions. Substitution of the native tyrosine with 3CNY yields a functional protein exhibiting hypsochromically shifted spectral properties. Time-dependent density functional theory (TD-DFT) calculations indicate that 3CNY incorporation results in a trans state at pH 8. Unlike mKate, the trans state is fluorescent. In contrast, incorporation of 3-chloro-L-tyrosine (3ClY) preserves the preference for the cis phenolate state. Molecular modelling suggests that the cyano group can form stabilising hydrogen bonds with residues S143 and S158, promoting the trans configuration. DFT analysis further indicates that the electron-withdrawing cyano group perturbs conjugation across the chromophore, potentially lowering the barrier to cis-trans isomerisation. Conversely, wild-type and 3ClY variants maintain polarised HOMO and LUMO distributions in the cis state, supporting stronger conjugation and a reduced HOMO-LUMO gap. Overall, the introduction of a genetically encoded 3-CNY tyrosine analogue into a fluorescent protein chromophore expands our mechanistic understanding and enables incorporation of a new chemical tag directly into the chromophore.

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.

We report a generalizable method for single-molecule localization super-resolution microscopy in living cells. Live-cell protein-based PAINT (points accumulation for imaging in nanoscale topography) is achieved by pairing exceptionally fluorogenic bis-trifluoromethyl rhodamine (BF) dyes with reversible HaloTag ligands. This far-red small molecule update to protein-based PAINT is readily incorporated into existing super-resolution microscopy workflows: pairing with photoactivatable fluorescent proteins further enables simultaneous two-color live-cell super-resolution microscopy and single-molecule diffusivity mapping (SMdM).

The incorporation of non-canonical amino acids (ncAAs) into proteins, developed in the past 20 years, has opened new avenues with respect to protein structure, protein modification, protein-protein interaction or enzyme catalysis beyond what is possible with the 20 proteinogenic AAs. Although >300 unusual building blocks including several ncAAs have been described in nonribosomal peptides (NRPs) naturally, we aimed to further expand the scope of the underlying nonribosomal peptide synthetases (NRPS) to incorporate ncAAs beyond the naturally available ones. We have therefore systematically screened for ncAA accepting NRPS systems, applied NRPS engineering to transfer the respective ncAA-accepting parts into other NRPSs and thereby created novel peptides that were further derivatized in post-enzymatic chemical synthesis reactions directly in bacterial culture extracts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/738027v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@90552forg.highwire.dtl.DTLVardef@1c8a5e0org.highwire.dtl.DTLVardef@2549dorg.highwire.dtl.DTLVardef@1012911_HPS_FORMAT_FIGEXP M_FIG C_FIG

Site-specific protein modification allows diverse functionalities to be introduced while minimizing perturbations to the protein structure and activity. Considerable efforts have been made to achieve site-specific modification of native proteins to overcome the heterogeneity resulting from conventional stochastic Lys or Cys modification. We have previously achieved the selective modification of Lys65 in a native immunoglobulin G1 (IgG1) antibody (trastuzumab) using EzMTG-pG(Fab), which is an engineered zymogen of microbial transglutaminase (EzMTG) fused to a Fab-binding protein G [pG(Fab)]. However, this approach cannot be widely applied to different types of IgG antibodies. Here, we designed pG(Fab)-EzMTG by fusing pG(Fab) to the N-terminus of EzMTG. Notably, switching the fusion partners dramatically altered the IgG modification site from Lys65 to Lys225, which is located in the hinge site of native IgG1 antibodies. This Lys225-selective labeling was applicable to different IgG1 antibodies. As a functional application, the cytotoxic drug monomethyl auristatin E (MMAE) was conjugated to Lys225 of trastuzumab, and the resulting antibody-drug conjugate exhibited antigen-specific cytotoxicity. These findings demonstrate that fusion-protein architecture determines site selectivity in proximity-directed enzymatic modification, providing a strategy for the site-specific functionalization of native antibodies.

Natural product synthesis by non-ribosomal peptide synthetases (NRPS) is greatly defined by the substrate selectivity of the adenylation (A) domains. Previous assays for specificity determination were mainly performed in vitro and were requiring protein purification. In this work, we developed - based on NRPS engineering - a novel in vivo assay suitable for high-throughput application named ASCR (A domain screening). Using the recently described XUT fusion sites, A domains and their upstream condensation domains were assembled as di-domains to characterized NRPS model system, which allowed detection of defined tripeptide products via mass spectrometry directly after cell culture extraction. We evaluated the assay by screening in total 54 A domains from five known and seven uncharacterized NRPS, covering a broad range organism taxonomy and GC content of the investigated NRPS-encoding genes. Additionally, we applied the assay to elucidate and confirm the structures of novel cyclic pentapeptides derived from three novel NRPS from Photorhabdus temperata K122.

O-GlcNAcylation is an important post translational modification that regulates numerous cellular processes, yet tools enabling selective removal of O GlcNAc from individual proteins via endogenous O-GlcNAcase (OGA) in living cells remain limited. Here, we report De-O GlcNAcylation-targeting chimeras (DOGTACs), a chemically induced proximity strategy that selectively reduces O GlcNAc from target proteins by recruiting endogenous OGA. Initial designs incorporating potent competitive OGA inhibitors efficiently engaged OGA but failed to induce de-O-GlcNAcylation, revealing that catalytic competence is essential for productive proximity-driven editing. By attenuating inhibitor potency while retaining sufficient OGA engagement, we developed optimized DOGTACs that promote concentration- and time-dependent, target-specific de-O-GlcNAcylation in living cells without perturbing global O-GlcNAc levels. Furthermore, we successfully applied DOGTAC to additional target proteins across multiple cell lines. Collectively, this work established attenuated competitive inhibitors as effective recruitment modules for catalytic enzyme engagement and a novel framework, DOGTAC, for targeted de-O-GlcNAcylation via endogenous OGA recruitment in living cells.

RNA-targeting small molecules are emerging as promising therapeutic modalities, but their development requires methods that define binding sites and evaluate RNA target selectivity. Existing approaches for detecting ligand-RNA interactions have provided powerful foundations, yet many rely on direct crosslinking or covalent-capture chemistries whose performance depends on ligand-specific probe design, warhead compatibility, and local reaction geometry. Here, we report Singlet Oxygen footprinting on RNA in a Ligand-Directed manner for Mutational Profiling (SOLiD-MaP), a photoproximity labelling platform for small molecule-RNA interaction analysis. Using the Mango-II aptamer and thiazole orange derivatives as a model system, we establish aniline as an efficient nucleophile for singlet oxygen-mediated RNA labelling and demonstrate target-selective labelling driven by ligand-localized photosensitization. We further show that labelling selectivity can be tuned by chemically constraining the singlet oxygen diffusion with a quencher. Finally, we develop a pairwise reverse transcription stop assay and a mutational profiling with next-generation sequencing readouts to infer ligand-proximal regions and unambiguously map binding sites. SOLiD-MaP provides a new, orthogonal strategy for studying small molecule-RNA recognition and should support RNA-focused mechanism-of-action studies.