The intermediate filament (IF) protein vimentin is a critical component of the cell cytoskeleton, yet our mechanistic understanding of head and tail domain function is incomplete. The C-terminal tail domain of vimentin is of increasing interest as it is essential for regulating the structure and mechanical properties of filament networks through interactions with divalent metal ions. However, the molecular basis of tail domain-metal interactions has not been characterized. Here, we analyze the structural and metal-binding properties of the vimentin tail domain. Mass spectrometry, UV-vis, and circular dichroism (CD) spectroscopy reveal binding of divalent copper (Cu(II)) to both a peptide composed of the last 11 residues of the tail domain and the complete, isolated tail domain. Solution nuclear magnetic resonance and CD measurements show that in the absence of Cu(II), the complete vimentin tail domain is primarily disordered and that Cu(II)-binding involves both the last 11 residues and another segment in the middle of the tail domain, leaving the N-terminal portion of the tail domain primarily disordered. Our study provides a Cu(II) binding model for the isolated vimentin tail domain that can assist with the interpretation of Cu(II) mediated effects on vimentin structure in biological contexts.
Guanosine- and deoxyguanosine-rich nucleic acids can form G-quadruplex structures (G4) that are stabilized by guanine tetrads (G4 tetrads). G4s find numerous applications in biotechnology. Here, we study a so called thrombin-binding aptamer (TBA), developed by SELEX procedures, that adopts a G4 conformation and inhibits clotting of thrombin. We investigate the TBA G4 and its variants with either four adenosine desoxynucleotides or four abasic sites attached either to the 5'-terminus (A4-TBA and ab4-TBA) or the 3'-terminus (TBA-ab4 and TBA-A4). These variants have been shown to exhibit differential anticlotting activities previously. The variant TBA-ab4, which was the most biological active in earlier investigations, has an exceptional stability against nuclease restriction, while all other variants show similar decay rates in mammalian serum. Biophysical characterization of the variants reveals that the structure of the aptamer remains unchanged, but that also their different thermal stabilities correlate with the anticlotting activity of TBA. Hydrogen exchange quantified by nuclear magnetic resonance spectroscopy (NMR) reveals individual G4 tetrad thermodynamics. Our data indicate that while enthalpy, entropy and free energy of base pair opening show surprisingly low variation, a hotspot for stabilization of the G4 is present at the 3', 5' terminal tetrad of TBA.
Peroxidation of polyunsaturated fatty acids in cellular membranes, when extensive and unrepaired, can lead to a form of eukaryotic cell death known as ferroptosis. A complex network of proteins and small molecules has evolved to modulate this peroxidation, thereby suppressing or enhancing ferroptosis. Within this network, the quinone reductase NQO1 has long been recognized for its ability to reduce and regenerate the membrane-resident antioxidant ubiquinone. Surprisingly, recent studies have also implicated NQO1 in pro-ferroptotic processes. Here, we present an experimental model designed to disentangle the opposing activities of NQO1 using a simple in vitro system composed of phospholipid liposomes. The biochemical setup enabled us to recapitulate the membrane association of the normally cytosolic NQO1 and to demonstrate that this association weakens in the presence of its cognate electron donor, NADH. The effect required the C-terminal tail of the enzyme and is likely linked to the higher disorder propensity of its last 50 amino acids. In the presence of NQO1, an increase in iron-driven liposome peroxidation was observed. Without quinone substrates in the system, these results support the idea that the NQO1 cofactor flavin adenine dinucleotide can reduce ferric (Fe3+) iron, thereby promoting reactive oxygen species generation and lipid peroxidation.
The membrane lipids of archaea differ from those of bacteria and eukarya in backbone stereochemical configuration, chemical linkage type, and hydrophobic chain structure, a fundamental dichotomy termed the lipid divide. Synthetic biology and metabolic engineering have enabled reconstruction of archaeal lipid biosynthesis pathways in bacterial and eukaryotic hosts, yielding production levels reaching 30% of membrane phospholipids in Escherichia coli and 6.5% of total cellular lipids in Saccharomyces cerevisiae. These achievements required coordinated expression of core archaeal enzymes combined with enhanced isoprenoid precursor supply. A recurring finding is that bacterial and eukaryotic enzymes exhibit remarkable substrate promiscuity toward archaeal lipid precursors, enabling biosynthesis of archaetidylglycerol, archaetidylethanolamine, and archaetidylinositol without requiring archaeal enzymes. This review examines the biosynthetic pathways, host systems, and engineering strategies underlying these advances. We consider how heterologous reconstitution informs longstanding questions about membrane evolution and the nature of the last universal common ancestor. Engineered strains with hybrid archaeal-bacterial membranes not only remain viable but also exhibit enhanced stress tolerance, demonstrating that the lipid divide does not preclude membrane coexistence while enabling biotechnological applications from stress-tolerant cell factories to archaeosome-based delivery systems.
Unspecific peroxygenase (UPO) is versatile fungal enzyme that carries out with high efficiency a plethora of C-H oxyfunctionalizations of organic compounds, simply triggered by H2O2. With the goal of making UPO an industrial biocatalyst, it has been subjected over the years to different directed evolution campaigns focused on expression, stability, activity, and selectivity. In this work, we tested the ability of PaDa-I and JaWa, evolved variants of the UPO from Agrocybe aegerita, to catalyze the oxidation of a series of pharmaceutical compounds, considered emerging contaminants (EC), or microcontaminants. Remarkably, these UPO mutants were able to catalyze the oxidation of 9 out of 12 EC, albeit with different efficacy. A detailed analysis of the reaction efficiency and the nontoxic nature of the reaction products point these UPO mutants as promising tools for enzymatic bioremediation schemes.
The effects on human health of chemical compounds, which might be present in the environment due to natural or anthropogenic causes, are a fundamental aspect to be considered for the protection of public health and workers' health and in the evaluation of industrial processes in terms of health protection and sustainability. Investigations focused on lesser known effects that have recently drawn more attention on potential human target proteins. Due to the complexity of biochemical interactions, it is not straightforward to determine the biological response resulting from exposure to a specific chemical. In this article, we tackle this issue by combining chemoinformatics tools and atomistic modeling to perform target identification for several compounds. The study was carried out using a publicly available database that collects relationships between chemicals, genes, and phenotypes and resulting diseases to validate the results of the target identification pipeline. Small molecules that may occur in occupational and nonoccupational settings were investigated. Finally, we discuss the potentialities and limitations of using these fast, computationally inexpensive methods in early-stage target identification, both with and without performing a literature search for experimental data.
Expanding strategies for the design of artificial protein dimers induced by metal ions is important for creating proteins with novel functions as well as useful research tools. In this study, we extended our previously established polyproline-based design and developed a method to induce dimerization suggestive of 3D domain swapping in a metal ion-dependent manner. Variants with six residues deleted from a loop in the C-terminal domain of outer surface protein A and containing His-Pro repeats formed dimers in the presence of divalent first-row transition metal ions. The formation and dissociation of the Zn2+-induced dimer occurred slowly, suggesting that dimerization requires substantial structural rearrangements. Moreover, the structure of the Zn2+-induced dimer predicted by AlphaFold3 was consistent with a 3D domain-swapped dimer stabilized by intermolecular coordination between Zn2+ and the histidine residues within the His-Pro repeats. This predicted structure remained stable during 100-ns molecular dynamics simulations. These experimental and computational evaluations suggest that the insertion of His-Pro repeats into loops is an effective strategy for designing metal ion-induced dimers suggestive of 3D domain-swapped dimers. Our results provide insights into expanding the design space of artificial metal ion-dependent protein dimers and advancing our understanding of the structural principles of metalloproteins.
Enantiopure cyanohydrins are versatile chiral intermediates of several bioactive molecules and life-saving drugs. Consequently, the demand for their efficient and sustainable preparation has grown significantly. We aimed to synthesise them using native and engineered Arabidopsis thaliana hydroxynitrile lyase (AtHNL). Screening of the in-house AtHNL variant library created by saturation mutagenesis at F179 and Y14 using Feigl-Anger paper-based assay towards the synthesis of four distinct industrially important chiral cyanohydrins has uncovered nine variants with more promising activity than the wild-type. After optimisation of eight crucial biocatalytic parameters using acetone cyanohydrin with the model reaction of asymmetric hydrocyanation of benzaldehyde, the substrate scope of the variants was studied by converting 21 different aromatic aldehydes, among which 18 are newly investigated by these enzymes, into their corresponding valuable enantiopure (R)-cyanohydrins with good yields (up to 98%) and excellent optical purities (up to >99.9%). Kinetic studies of AtHNL variants revealed significant improvements, with a >27-fold increase in catalytic efficiency over the wild-type in the hydrocyanation of 4-allyloxybenzaldehyde. The improved catalytic performance was supported by molecular docking and simulation studies. These findings highlight the potential of AtHNL variants in enhancing activity, synthetic scope, and catalytic efficiency towards sustainable synthesis of industrially relevant chiral cyanohydrins.
Fluorescent probes have been widely developed for detecting amyloid biomarkers associated with neurodegenerative diseases (NDs). The requirement for external light for excitation, however, limits their utility for research and in vivo applications. Chemiluminescent probes, which use chemical reactions to generate emissive excited states rather than externally applied light, may offer a promising alternative to overcome some limitations for optical imaging of amyloid biomarkers. Here, we designed and synthesized a chemiluminescent amyloid-binding probe (CLIP-1) and evaluated its capability to label three amyloid biomarkers for NDs-amyloid β (Aβ), α-synuclein (α-syn), and tau protein aggregates. We found that CLIP-1 exhibits markedly enhanced chemiluminescence (CL) in aqueous solution when activated by ambient oxygen in the presence of aggregated Aβ, whereas little to no emission is observed in the absence of aggregates or in the presence of monomeric Aβ. Additionally, CLIP-1 displayed a different wavelength of emission when bound to tau aggregates compared to Aβ or α-syn aggregates, which we attribute to differences in the relative overall charge of the amyloids at neutral pH. Imaging of brain slices confirmed enhanced CL of CLIP-1 in an Alzheimer's disease mouse model, highlighting the potential for amyloid-targeting chemiluminescent probes as new tools for aiding in diagnosis of amyloid-associated neurodegenerative diseases.
Unbiased atomistic molecular dynamics simulations have been employed to examine the binding interactions between amyloid fibrils (Aβ(1-42) and tau) with various ligands: anionic pFTAA, qFTAA-CN, neutral HS-276, and cationic bTVBT4. The ligand-fibril interactions were analyzed in a two-step approach. First, an analysis of the spatial distributions of the ligands around the amyloid fibril protofilaments was carried out to identify prospective binding sites. Second, the associated ligand-fibril binding energies at these sites were determined using umbrella sampling. The results reveal that pFTAA and qFTAA-CN share common binding sites in both Aβ(1-42) and tau fibrils. Ligands bTVBT4 and HS-276, on the other hand, are found to have different binding sites in Aβ(1-42) but share the same site in tau fibrils. An analysis of the spatial distributions of all ligand-fibril pair combinations under comparable conditions shows the lowest densities when bTVBT4 interacts with Aβ(1-42) and HS-276 with the tau fibril. These findings are corroborated by fluorescence costaining experiments, in which bTVBT4 and HS-276 show no correlation with Aβ-specific and tau-specific antibody markers, respectively. Further structural analysis reveals significant changes in the conformations of all ligands upon binding to the proteins compared to their conformations in aqueous solution. The binding of the anionic ligands (multiple localized negative charges) to fibrils is primarily driven by Coulombic forces, whereas the binding of the neutral HS-276 and cationic bTVBT4 (single delocalized positive charge) is governed by Lennard-Jones interactions. This highlights the influence of charges on binding, providing insights for the design of future ligands targeting Aβ(1-42) and tau fibrils.
Antimicrobial resistance (AMR) has emerged as a global healthcare crisis, necessitating the discovery of potent antibacterial agents. In the present investigation, we report the discovery, in vitro and ex vivo antibacterial efficacy studies, and mechanism of action of an unexplored benzimidazole derivative (BI-10), a promising broad-spectrum antibacterial agent with significant efficacy against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). BI-10 demonstrated a minimum Inhibitory Concentration (MIC) of 2.4 μg/ml (6.25 µM) against these priority pathogens. In vitro assessments revealed that BI-10 possessed rapid bactericidal activity, anti-biofilm potential and showed synergistic interactions with conventional antibiotics. Ex vivo efficacy studies using various mammalian cell lines demonstrated strong intracellular killing. Moreover, BI-10 showed the ability to prevent both adhesion and invasion of pathogens in different mammalian cell infection models. The membrane-disrupting nature of BI-10 against both pathogens was established using scanning electron microscopy, membrane permeability, depolarization, and integrity assays. Multiple in silico analyses further demonstrated drug-likeness and the biocompatibility profile of BI-10. Altogether, our findings establish BI-10 as a potent, broad-spectrum antimicrobial agent with robust in vitro and ex vivo efficacy and bacterial cell membrane-disrupting activity. This study highlights BI-10 as a valuable antibacterial lead molecule for the development of a new antimicrobial agent against multidrug-resistant pathogens.
S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) are generally classified as C-, O-, N-, S-, or halide MTs depending on their methyl acceptor. C-MTs catalyze selective methylation reactions of carbon nucleophiles and play a crucial role in the regulation and diversification of natural products. The control of chemoselectivity by these enzymes is poorly understood, especially with respect to the resonance of a nucleophilic neighboring group that activates the carbon methylation site. We investigated two aromatic C-MTs for the underlying mechanisms governing their chemo- and/or regioselectivity. The unprecedented in vitro dimethylation activity of SfmM2 and NapB5 was demonstrated using the native substrate l-tyrosine and substrates with a 2,4-dihydroxyacetophenone pattern, respectively. Substrate symmetry and the in situ SAM supply with removal of the competitive inhibitor S-adenosyl-l-homocysteine are favorable for dimethylation activity. Through NapB5 catalysis, we obtained C-(di-)methylated acetylphloroglucinol and flavonoid derivatives. We discovered that NapB5 catalyzes both C- and O-methylation of sterically demanding flavonoids. Here, chemoselectivity was modulated by the geometry of substrate binding through substrate selection or site-directed mutagenesis. Precise positioning of the acceptor nucleophile toward SAM is required to achieve regio- and chemoselectivity despite competing C- and O-nucleophilic sites. Thus, chemoselectivity is context-dependent, which opens new horizons for the diversification of natural products.
This study presents a novel multienzymatic strategy for the efficient and green production of valuable salicylic acid from plant-derived salicin. The biocatalytic route couples β-glucosidase for deglycosylation with laccase and xanthine oxidase (XO) for subsequent oxidation. The primary objective of this approach was to assess the feasibility of enzyme coupling within a single one-pot reaction. The results demonstrate that this strategy successfully enabled the transformation of salicylic alcohol into salicylic acid, mediated by a compatible laccase/TEMPO and XO system in a one-step process at pH 7 and 25°C. However, the integration of β-glucosidase into a one-pot system for direct salicin conversion was found to reduce the oxidative efficiency of XO, thus necessitating a sequential approach. Applying this optimized enzymatic cascade to a crude Salix purpurea bark extract initially encountered limitations in oxidation due to matrix complexity. Subsequent optimization of biocatalyst dosage delivered complete substrate oxidation and a 95% salicylic acid yield. This research significantly broadens the applicability of multienzymatic biocatalytic cascades, offering a promising and sustainable route for transforming natural compounds within complex plant extracts into high-value bioactive ingredients.
Indocyanine green (ICG) is a clinically approved fluorescent dye that is used for lesion identification during hepatobiliary surgery. However, the chemical structure of ICG seems to be suboptimal for this excretion-based diagnostic readout. In this study the effect of systematic structural variations in Cy5-analogs were evaluated to elucidate the structure-activity relationship between hepatic uptake and excretion. Nine Cy5 dyes with variations in N-alkyl indole substitution (butyl sulfonate or methyl), and benzoannulation were synthesized in analogy to the ICG scaffold (Cy7.5-(SO3-)2). Photo-, bio- and chemical properties were analyzed and combined to identify structure-activity relationships using Spearman's correlation and multiparametric analysis. Chemical modifications were shown to alter (photo)chemical properties and constituted in clear biological effects; Sulfonate substitution supports hepatic excretion, while benzoannulation promoted nonspecific background accumulation. Cy5-(SO3-)2, rather than the ICG-analog Cy5.5-(SO3-)2, was identified as lead. Systematic evaluation revealed key structural determinants that influence biliary excretion and allowed lead selection of dyes for hepatobiliary imaging. These insights provide a foundation for the rational design of optimized fluorescent agents for this application.
Autoimmune diseases are conditions characterized by aberrant B-cell and T-cell reactivity against self-antigens. Autoantibodies are serological biomarkers of autoimmune diseases, as such, autoantibody testing is a key step for diagnosing and classifying many autoimmune diseases, as well as monitoring disease activity and devising a treatment strategy. Considering the rising number of people affected by autoimmune diseases worldwide, it is even more important to have efficient techniques that combine high sensitivity and specificity with reduced sample processing times and an automated high-throughput workflow. In this context, the identification and validation of new autoantigens and autoantibodies, together with the implementation of technological advancements, has led, in the last decades, to an improvement in patient diagnosis and stratification. Here, we review the major antigens of some of the most common autoimmune diseases, and the most widely used assays employed in diagnostic laboratories for the detection of their cognate antibody, confronting more traditional platforms with emerging ones in selected cases of study.
Adenosine vinylsulfonamide (AVS) probes provide a powerful means of stabilizing the thioesterification state in nonribosomal peptide synthetases (NRPSs) by mimicking aminoacyl-AMP intermediates. Existing synthetic strategies have enabled broad application of this probe class, yet they often rely on strongly basic conditions for constructing the key CC bond from α-amino aldehydes, substrates that are inherently prone to epimerization. These features limit practical access to AVS derivatives from nonproteinogenic amino acids, many of which are central to NRPS-mediated natural product biosynthesis. To address these constraints, we developed a concise three-step route beginning from N-Boc amino alcohols that proceeds under operationally simple and stereochemically controlled conditions. A preassembled adenosine phosphinyl sulfonamide serves as an effective coupling partner in a Ba(OH)2-mediated Horner reaction performed near 0°C, enabling highly diastereoselective AVS formation with minimal epimerization under mild, open-air conditions. The method tolerates diverse amino acid-derived substrates, including electron-rich L-Tyr derivatives relevant to tetrahydroisoquinoline alkaloid biosynthesis. Functional evaluation by LC-MS/MS confirmed covalent capture of the NRPS SfmC by an L-Tyr-derived probe, demonstrating that AVS constructs prepared by this route retain full biochemical competence. This streamlined synthesis enhances reliable access to stereochemically well-defined AVS probes and supports structural and mechanistic studies across diverse NRPS systems.
Adenylation enzymes transfer acyl substrates selectively onto carrier proteins (CPs) in natural product biosynthesis. Despite the importance of adenylation enzyme-CP interactions, structural information on these transient complexes remains limited. Previously, we developed a pantetheine cross-linking probe (named C2Br), which contains an ethylenediamine linker with a reactive bromoacetamide group, and determined the structure of the cross-linked complex of the adenylation enzyme HitB with the CP HitD. Here, we investigated the linker-length effects of pantetheine probes in the cross-linking reactions of two adenylation enzymes, HitB and EntE, with CPs using probes with different diamine linkers, such as C2Br and C4Br, the latter containing a longer butanediamine linker moiety. Both adenylation enzymes formed cross-linked complexes with CPs irrespective of the probe used, but the reaction efficiencies depended on the linker length. Crystal structural analysis showed that the HitB-HitD interface interactions in the HitB-C4Br-HitD complex are essentially identical to those in the HitB-C2Br-HitD complex. In contrast, the diamine moieties of probes adopt different interaction modes, accounting for the observed variations in cross-linking efficiencies. A repertoire of pantetheine probes with varying linker lengths will facilitate structural studies on adenylation enzyme-CP interactions by enabling optimization for each adenylation enzyme.
Gastrointestinal systems of mammals and birds host taxonomically complex and functionally diverse microbial communities. Microbial activities contribute to community functioning and interaction with the host but can also be exploited as a source of novel enzymes or other industrially relevant microbial traits. With the overall goal to identify new resources for carbohydrate-active enzymes (CAZymes), we bioprospected fecal microbial communities of the little-explored source of captive wild animals. Using dbcan3, we identified a CAZyome dominated by glycosyl hydrolases (GHs) specialized in degrading oligo- and polysaccharides with much lower diversity and abundance of glycosyl transferases, carboxyl esterases, polysaccharide lyases, and redox enzymes with auxiliary activity. CAZyome profiles differed between animals depending on gut physiology and diet. Crude cell extracts conferred hydrolytic activity against compositionally and structurally diverse polysaccharides and nitrophenyl-sugar analogs. We identified five candidate GH68 and GH70 enzymes with the potential to produce oligo- and polysaccharides from sucrose, highlighting that fecal metagenomes are a source of rare CAZymes with industrial relevance. Taken together, we exemplify the functional potential captive wild animal fecal microbiota and suggest such a gene pool as a largely untapped resource for the discovery of novel biotechnological applications.
The relationship between conformational dynamics and multistate transition kinetics has been the fundamental focus for understanding the DNA Holliday junction (HJ) intermediated homologous genetic recombination process. Although the crystal structure and Markovian dynamics of HJ are known, the nature of the dynamics related to the transition frequency to thermodynamically stable multistate and intermediate states is not yet fully understood. Using single-molecule fluorescence resonance energy transfer (smFRET) and correlation based statistical analysis, we have identified intermittent conformational dynamics with three different patterns: periodic oscillation, correlated stochastic fluctuation, and damped oscillation. Furthermore, we found that the intermittently correlated stochastic fluctuation conformational dynamics of HJ goes through multistep transition kinetics along the transition coordinates, presenting an extreme pattern of conformational dynamics. There is also heterogeneity in the correlated stochastic fluctuation frequency due to heterogeneity in the molecular dynamics. Our results on conformational dynamics of HJ also support the multistate transition model. We applied multipoint time-correlation function analysis to smFRET efficiency time-trajectories resulting from Cy3/Cy5 labeled HJ to determine and compare the kinetics of various possible multistate conformational pathways. Results from the time correlation function (TCF) analysis show that intermediate conformational states between the energy states of two extreme conformations likely play a critical role in loop-gated conformational change mechanisms in HJ-mediated genetic recombination processes.
Natural products containing vinylogous amino acids are rarely found in nature and often possess significant biological activity. Barnesin A was the first NP reported from an anaerobic bacterium (Sulfurospirillum barnesii) postulated to be biosynthesized by a nonribosomal peptide synthetase (NRPS) polyketide synthases (PKS) hybrid. Containing a vinylogous arginine moiety, the lipodipeptide exhibited nanomolar inhibitory activity against cysteine proteases. While a putative NRPS-PKS hybrid biosynthetic gene cluster (brn) was identified and a trans-acting acyltransferase (trans-AT) domain was postulated, experimental validation remained an open question. Here, we report the production of barnesin A by heterologous expression of the trans-AT domain-dependent NRPS-PKS gene cluster in Escherichia coli. Our findings indicate that the native primary metabolism-derived malonyl CoA-acyl carrier protein transacylase homolog (FabD) functions as a trans-AT in the biosynthesis pathway, while the NRPS-PKS megaenzyme exhibited strict selectivity toward its native phosphopantetheinyl transferase. Metabolome mining further allowed for the description of previously unreported barnesin congeners. The results of this study enabled the establishment of a biosynthetic platform for the generation of novel lipopeptidic vinylogous protease inhibitors.