ACS Chemical Biology最新文献

Significant effort has been directed toward the characterization of nonheme iron enzymes owing to their breadth of unique reactivity. Through genome mining, we identified a conserved biosynthetic gene cluster within Pseudomonadota encoding one such family, the multinuclear nonheme iron-dependent oxidative enzymes (MNIO, formerly DUF692). Using a representative gene cluster from Fontimonas thermophila, we heterologously produced the post-translationally modified peptide fontiphorin, and detailed spectral analysis revealed MNIO-catalyzed installation of seven 5-thiooxazole (5TO) moieties. During our work, additional MNIO products were reported with conflicting structural assignments, so we investigated the related biosynthetic gene clusters from Haemophilus influenzae and Neisseria gonorrhoeae. Using alkylation-assisted HMBC correlations, we demonstrated that these products also contain 5TO, resulting in a revision of the structure of oxazolin. We further provide evidence supporting a role for 5TO-containing peptides in copper detoxification and recommend that this emerging class of Cu-associated peptidic thiooxazole metallophores be referred to as captophorins. To further explore the captophorins, we reconstituted fontiphorin biosynthesis in vitro and investigated its enzymatic requirements. Using cell-free production of single-site, double-site, and naturally occurring variants, we examined enzyme-substrate interactions to determine key sites governing catalysis by 5TO-forming MNIOs. Through our detailed spectroscopic approach for 5TO assignment and investigation of enzyme-substrate interactions, our work unifies tens of thousands of MNIOs in the biosynthesis of captophorins.

Bacterial membrane vesicles (MVs) are natural delivery systems for biomolecules, such as enzymes and nucleic acids, but their role in transporting specialized metabolites is less understood. Many microbial metabolites are lipophilic and poorly water-soluble, raising questions about how they perform ecological functions in aquatic environments. Here, we demonstrate that Pseudoalteromonas piscicida JC3, a marine bacterium with probiotic potential, packages lipophilic depsipeptides known as bromoalterochromides (BACs) into outer membrane vesicles. Untargeted metabolomics and molecular networking identified six known and two previously unknown BACs, while targeted LC-MS/MS localized BACs to MVs and cells, with no detection in culture supernatants. Structure elucidation of a new analogue, bromoalterochromide E/E', was achieved through isolation and spectroscopic analysis, including modified Marfey's analysis to determine amino acid composition and chirality. Functional assays showed that BAC-loaded MVs exhibit antibacterial activity against Staphylococcus aureus and the marine pathogen Vibrio anguillarum, linking vesicle-mediated metabolite delivery to microbial competition. These findings highlight MVs as transporters of lipophilic natural products and suggest their potential as natural drug delivery vehicles in clinical and aquaculture settings.

Efficient delivery remains a major challenge for therapeutic genome editing because many widely used CRISPR nucleases are large and leave limited space for regulatory elements or additional payloads in a single adeno-associated virus (AAV) vector. Miniature Cas12 nucleases are particularly appealing, as their reduced size alleviates packaging constraints while preserving RNA-guided DNA cleavage. Here, we outline a workflow that links large-scale sequence mining with phylogenetic and structural filtering, followed by PAM profiling, in vitro cleavage, bacterial genome interference, and genome-editing assays in human cells to confirm activity. This protocol is intended to distill broad sequence collections into a small set of compact Cas12 nucleases with demonstrated functions that can serve as starting points for further engineering in delivery-limited settings.

Quantitative live cell monitoring of catalytic activity is essential for advancing chemical biology, yet designing substrate probes that combine broad applicability with finely tunable kinetics remains a significant challenge. While glyco-bisacetal-based substrates (BABS) have proven applicable to several enzymes, their alkyl-hemiacetal core can limit turnover rates for certain enzymes. Herein, we report a novel one-pot, three-component glycosylation strategy to synthesize Aryl-BABS through the trapping of transient aryl-hemiacetals. This approach enables rapid diversification of the bisacetal scaffold using various phenols, yielding a library of aryl-bisacetal substrates. Kinetic evaluation of catalytic hydrolysis with a model glycosidase demonstrated that these Aryl-BABS are efficiently processed, with turnover rates up to 2 orders of magnitude faster than analogous alkyl glycosides and approaching those seen for activated p-nitrophenyl glycosides. Simple substitutions to phenol lead to a 20-fold range of kinetic tunability. Crucially, stopped-flow studies combined with kinetic simulations revealed that the breakdown of the enzymatically released aryl-hemiacetal is extremely rapid, at least 100-fold faster than that of alkyl-hemiacetals. This synthetic and kinetic tunability offers a powerful roadmap for developing advanced substrate probes of biocatalysts, eventually enabling quantitative measurement of previously intractable enzymes in living systems.

Riboglow probes are small molecules where a synthetic fluorophore is connected to an RNA-binding moiety via a chemical linker. Upon binding a short RNA sequence, probe fluorescence intensity and lifetime increase. The fluorescence change is modulated by the architecture of the chemical linker. Here, we systematically interrogated the linker composition in a series of Riboglow probes and assessed fluorescence properties. We found that glycine linkers result in higher fluorescence turn-on compared to a polyethylene glycol linker of similar length. When varying the length of the polyglycine linker, we found that increasing the number of glycine residues led to more substantial fluorescence turn-on upon RNA-ligand binding. Surprisingly, the composition of the Riboglow chemical linker influences fluorescence lifetime contrast when comparing probe binding to two different RNA ligands, a quality necessary for RNA multiplexing. Finally, evaluating probe fluorescence lifetimes in live mammalian cells demonstrated the ability of new Riboglow probes to visualize RNAs live. Insights gained from the systematic assessment of the linker’s architecture will dictate the rational design of future fluorophore-quencher probe designs.

Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. Micromonospora echinospora subsp. challisensis NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens, including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety; yet, the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain M. echinospora ATCC 15837 that contains both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (tln1 and tln5) that putatively encode pyridoxal 5′-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography–mass spectrometry (LC–MS) identified l-alanine, l-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective β-substitution reaction of O-phospho-l-serine with a preferential d-alanine nucleophile. Alanine racemization and pseudodipeptide l-serine-Cβ-N-d-alanine (d,l-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics.

Bacteria use a process of chemical communication called quorum sensing to regulate group behaviors. Quorum sensing relies on the synthesis, release, and detection of signal molecules called autoinducers that accumulate with increasing cell density. The pathogen Vibrio cholerae makes and detects three autoinducers which together, regulate genes required for group behaviors including virulence and biofilm formation. Two autoinducers are produced by dedicated autoinducer synthases that employ S-adenosyl methionine as a substrate. The third autoinducer, 3,5-dimethylpyrazin-2-ol (DPO), is produced from threonine and alanine. The threonine dehydrogenase (Tdh) enzyme oxidizes l-threonine to 2-amino-3-ketobutyric acid, which spontaneously decarboxylates to aminoacetone. Here, we define the steps required to convert aminoacetone and alanine into DPO. We show that diverse adenylate-forming enzymes can condense ATP and d- or l-alanine to form alanyl-adenylate, the necessary intermediate in DPO biosynthesis. Upon release, alanyl-adenylate spontaneously condenses with aminoacetone to form N-alanyl-aminoacetone, which cyclizes to form DPO. We propose that DPO is distinct from other autoinducers in that there is apparently no dedicated synthase. Rather, a collection of enzymes contribute to the production of this quorum-sensing autoinducer.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron loss. ALS-linked mutations in UBQLN2 promote protein aggregation and disrupt proteostasis, yet the mutation-specific protein interactomes and their functional relevance remain poorly defined. We employed APEX2 proximity labeling, together with affinity enrichment of biotinylated peptides and LC–MS/MS analysis, to profile the interactomes of wild-type UBQLN2 and two ALS-linked variants, UBQLN2^P497H and UBQLN2^P497S. We identified 785 unique biotinylated proteins, many of which exhibit augmented enrichment in the proximity proteomes of the two mutants over wild-type UBQLN2. Notably, the E3 ubiquitin ligases TRIM9 and TRIM26 were selectively enriched in the proximity proteome of UBQLN2^P497H, which we validated by coimmunoprecipitation followed by Western blot analysis. Fractionation analysis revealed coaccumulation of TRIM9 and TRIM26 with UBQLN2^P497H in the insoluble fraction, consistent with its heightened aggregation propensity. Treatment of UBQLN2^P497H-expressing cells with a proteasomal inhibitor led to elevated accumulation of a C-terminal UBQLN2 fragment that is absent in cells expressing wild-type UBQLN2 or its P497S mutant. Individual knockdown of TRIM9 and TRIM26 significantly increased the abundance of the fragment, establishing UBQLN2^P497H as a substrate for TRIM9- and TRIM26-mediated ubiquitinylation and subsequent proteasomal degradation. These findings nominate TRIM9 and TRIM26 as specific interactors of UBQLN2^P497H and as regulators of a previously underexplored C-terminal UBQLN2 fragment, suggesting that impaired clearance of this species may contribute to ALS pathogenesis.

The epidermal growth factor receptor (EGFR) mediates signal transduction by triggering downstream phosphorylation to regulate cell proliferation. However, the complexity of the cellular environment has limited in situ structural investigations of membrane proteins within their native context. Here, we present a proof-of-concept study integrating protein cage labeling with cryo-electron tomography (cryo-ET) to directly visualize receptor assemblies on the native membrane. Using EGFR as a model system, we demonstrate that the protein cage can associate with multiple EGFR molecules, thereby inducing their oligomerization. The distance between neighboring EGFRs within these assemblies was measured to be 7.1 ± 1.2 nm. Furthermore, we validated the functional relevance of this system by showing that protein cage-induced EGFR assemblies were accompanied by enhanced ligand-independent phosphorylation. In summary, our results establish the feasibility of using protein cage-labeling for the induction and in situ structural analysis of membrane protein oligomerization.

The oncogene MYCN is predominantly expressed in cancer stem-like cells, where it drives tumor growth, metastasis, and therapeutic resistance in hepatocellular carcinoma (HCC). In this study, we explored MYCN Inhibitors (MI) from the RIKEN Natural Products Depository chemical library and identified NPD15261 (designated as MI102) as a selective small-molecule inhibitor of MYCN expression. MI102 markedly reduced MYCN mRNA and protein levels in HCC cells, suppressing proliferation and colony formation, while inducing apoptosis, with minimal impact on normal hepatic cells. Mechanistically, kinase profiling revealed that MI102 is a highly selective inhibitor of MET receptor tyrosine kinase that specifically blocks phosphorylation at Y1234/Y1235. Hepatocyte growth factor-mediated MET activation induces MYCN expression and partially rescues MI102-mediated MYCN suppression. Notably, MI102 effect exhibited superior tumor cell selectivity compared with the MET inhibitor tivantinib. At the transcriptional level, RNA-seq revealed that MI102 globally downregulated MYCN-associated oncogenic programs. Collectively, these findings establish pharmacological downregulation of MYCN as a promising therapeutic strategy for HCC and reveal a functional link between MET signaling and MYCN-driven oncogenic pathways.