Search Results (968)

The barbel chub (Squaliobarbus curriculus) exhibits remarkable resistance to grass carp reovirus (GCRV), a devastating pathogen in aquaculture. To reveal the molecular basis of this resistance, we investigated complement factor D (DF)—a rate-limiting serine protease governing alternative complement pathway activation. Molecular cloning revealed that the barbel chub DF (ScDF) gene encodes a 1251-bp cDNA sequence translating into a 250-amino acid protein. Crucially, bioinformatic characterization identified a unique N-glycosylation site at Asn¹³⁹ in ScDF, representing a structural divergence absent in grass carp (Ctenopharyngodon idella) DF (CiDF). While retaining a conserved Tryp_SPc domain harboring the catalytic triad (His⁶¹, Asp¹⁰⁹, and Ser²⁰⁴) and substrate-binding residues (Asp¹⁹⁸, Ser²¹⁹, and Gly²²¹), sequence and phylogenetic analyses confirmed ScDF’s evolutionary conservation, displaying 94.4% amino acid identity with CiDF and clustering within the Cyprinidae. Expression profiling revealed constitutive ScDF dominance in the liver, and secondary prominence was observed in the heart. Upon GCRV challenge in S. curriculus kidney (SCK) cells, ScDF transcription surged to a 438-fold increase versus uninfected controls at 6 h post-infection (hpi; p < 0.001)—significantly preceding the 168-hpi response peak documented for CiDF in grass carp. Functional validation showed that ScDF overexpression suppressed key viral capsid genes (VP2, VP5, and VP7) and upregulated the interferon regulator IRF9. Moreover, recombinant ScDF protein incubation induced interferon pathway genes and complement C3 expression. Collectively, ScDF’s rapid early induction (peaking at 6 hpi) and multi-pathway coordination may contribute to barbel chub’s GCRV resistance. These findings may provide molecular insights into the barbel chub’s high GCRV resistance compared to grass carp and novel perspectives for anti-GCRV breeding strategies in fish. Full article

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of cell wall-associated enzymes involved in the construction and remodeling of cellulose/xyloglucan crosslinks. However, knowledge of this gene family in the model monocot Brachypodium distachyon is limited. A total of 29 BdXTH genes were identified from the whole genome, and these were further divided into three subgroups (Group I/II, Group III, and the Ancestral Group) through evolutionary analysis. Gene structure and protein motif analyses indicate that closely clustered BdXTH genes are relatively conserved within each group. A highly conserved amino acid domain (DEIDFEFLG) responsible for catalytic activity was identified in all BdXTH proteins. We detected three pairs of segmentally duplicated BdXTH genes and five groups of tandemly duplicated BdXTH genes, which played vital roles in the expansion of the BdXTH gene family. Cis-elements related to hormones, growth, and abiotic stress responses were identified in the promoters of each BdXTH gene, and when roots were treated with two abiotic stresses (salinity and drought) and four plant hormones (IAA, auxin; GA3, gibberellin; ABA, abscisic acid; and BR, brassinolide), the expression levels of many BdXTH genes changed significantly. Transcriptional analyses of the BdXTH genes in 38 tissue samples from the publicly available RNA-seq data indicated that most BdXTH genes have distinct expression patterns in different tissues and at different growth stages. Overexpressing the BdXTH27 gene in Brachypodium led to reduced root length in transgenic plants, which exhibited higher cellulose levels but lower hemicellulose levels compared to wild-type plants. Our results provide valuable information for further elucidation of the biological functions of BdXTH genes in the model grass B. distachyon. Full article

Class D β-lactamases (DBLs) represent a major threat to antibiotic efficacy by hydrolyzing β-lactam drugs, including last-resort carbapenems, thereby driving antimicrobial resistance in Gram-negative bacteria. The enzymes share a structurally conserved two-domain α/β architecture with seven active-site motifs and three flexible extended loops (the P-loop, Ω-loop, and newly designated B-loop) that surround the active site. While each of these loops is known to influence enzyme function, their coordinated roles have not been fully elucidated. To investigate the significance of their interplay, we compared the sequences and crystal structures of 40 DBLs from clinically relevant Gram-negative pathogens and performed molecular dynamics simulations on selected representatives. Combined structural and dynamical analyses revealed a strong correlation between B-loop architecture and carbapenemase activity in the pathogens Klebsiella and Acinetobacter, particularly regarding loop length and spatial organization. These findings emphasize the B-loop’s critical contribution, in concert with the P- and Ω-loops, in tuning active site versatility, substrate recognition, catalytic activity, and structural stability. A deeper understanding of how these motifs and loops govern DBL function may inform the development of novel antibiotics and inhibitors targeting this class of enzymes. Full article

Candidozyma auris is a multidrug-resistant, thermo- and osmotolerant yeast capable of persisting on biotic and abiotic surfaces, attributes likely linked to its cell wall composition. Here, seven putative genes encoding yapsins, aspartyl proteases GPI-anchored to the membrane or cell wall, were identified in the genomes of C. auris CJ97 and 20-1498, from clades III and IV, respectively. The C. auris YPS1 gene is orthologous to the SAP9 of C. albicans. The YPS7 gene is orthologous to YPS7 in C. glabrata and S. cerevisiae, so that they may share similar roles. An in silico analysis suggested an interaction between pepstatin and the catalytic domain of Yps1 and Yps7. Although this inhibitor, when combined with caffeine, had a subtle effect on the growth of C. auris, it induced alterations in the cell wall. CauYPS1 and CauYPS7 expression increased under nutrient starvation and NaCl, and at 42 °C. The transcriptome of the 20-1498 strain suggests that autophagy may play a role in thermal stress, probably degrading deleterious proteins or maintaining cell wall and vacuolar homeostasis. Therefore, CauYps1 and CauYps7 may play a role in the cell wall integrity of C. auris in stress conditions, and they could be a target of new antifungal or antivirulence agents. Full article

The shikimate pathway is the fundamental metabolic route for aromatic amino acid biosynthesis in bacteria, plants, and fungi, but is absent in mammals. This review explores how multi-enzyme synergy and allosteric regulation coordinate metabolic flux through this pathway by focusing on three key enzymes: 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, chorismate mutase, and tryptophan synthase. We examine the structural diversity and distribution of these enzymes across evolutionary domains, highlighting conserved catalytic mechanisms alongside species-specific regulatory adaptations. The review covers directed evolution strategies that have transformed naturally regulated enzymes into standalone biocatalysts with enhanced activity and expanded substrate scope, enabling synthesis of non-canonical amino acids and complex organic molecules. Industrial applications demonstrate the pathway’s potential for sustainable production of pharmaceuticals, polymer precursors, and specialty chemicals through engineered microbial platforms. Additionally, we discuss the therapeutic potential of inhibitors targeting pathogenic organisms, particularly their mechanisms of action and antimicrobial efficacy. This comprehensive review establishes the shikimate pathway as a paradigmatic system where understanding allosteric networks enables the rational design of biocatalytic platforms, providing blueprints for biotechnological innovation and demonstrating how evolutionary constraints can be overcome through protein engineering to create superior industrial biocatalysts. Full article

An anion exchange-assisted technique was used for the synthesis of platinum-decorated SnO₂ supports, providing nanocatalysts with enhanced activity for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). In this study, a series of SnO₂ supports, namely SnA (synthesized almost at room temperature), SnB (hydrothermally treated at 180 °C), and SnC (annealed at 600 °C), are systematically investigated, all loaded with 1 mol% Pt from H₂PtCl₆ under identical mild conditions. The chloride ions from the SnCl₄ precursors were efficiently removed via a strong-base anion exchange reaction, resulting in highly dispersed, crystalline ~5 nm cassiterite SnO₂ particles. All Pt/SnO₂ composites displayed mesoporous structures with type IVa isotherms and H₂-type hysteresis, with SP1a (Pt on SnA) exhibiting the largest surface area (122.6 m²/g) and the smallest pores (~3.5 nm). STEM-HAADF imaging revealed well-dispersed PtOx domains (~0.85 nm), while XPS confirmed the dominant Pt⁴⁺ and Pt²⁺ species, with ~25% Pt⁰ likely resulting from photoreduction and/or interactions with Sn–OH surface groups. Raman spectroscopy revealed three new bands (260–360 cm⁻¹) that were clearly visible in the sample with 10 mol% Pt and were due to the vibrational modes of the PtOx species and Pt-Cl bonds introduced due the addition and hydrolysis of H₂PtCl₆ precursor. TGA/DSC analysis revealed the highest mass loss for SP1a (~7.3%), confirming the strong hydration of the PtOx domains. Despite the predominance of oxidized PtOx species, SP1a exhibited the highest catalytic activity (k_app = 1.27 × 10⁻² s⁻¹) and retained 84.5% activity for the reduction of 4-NP to 4-AP after 10 cycles. This chloride-free low-temperature synthesis route offers a promising and generalizable strategy for the preparation of noble metal-based nanocatalysts on oxide supports with high catalytic activity and reusability. Full article

Large-conductance, voltage- and calcium-activated potassium (BK) channels are crucial regulators of cellular excitability, influenced by various signaling molecules, including heme. The BK channel contains a heme-sensitive motif located at the sequence ⁶¹²CKACH⁶¹⁶, which is a conserved heme regulatory motif (HRM) found in the cytochrome c protein family. This motif is situated within a linker region of approximately 120 residues that connect the RCK1 and RCK2 domains, and it also includes terminal α-helices similar to those found in cytochrome c family proteins. However, much of this region has yet to be structurally defined. We conducted a sequence alignment of the BK linker region with mitochondrial cytochrome c and cytochrome c domains from various hemoproteins to better understand this functionally significant region. In addition to the HRM motif, we discovered that important structural and functional elements of cytochrome c proteins are conserved in the BK RCK1-RCK2 linker. Firstly, the part of the BK region that is resolved in available atomic structures shows similarities in secondary structural elements with cytochrome c domain proteins. Secondly, the Met80 residue in cytochrome c domains, which acts as the second axial ligand to the heme iron, aligns with the BK channel. Beyond its role in electron shuttling, cytochrome c domains exhibit various catalytic properties, including peroxidase activity—specifically, the oxidation of suitable substrates using peroxides. Our findings reveal that the linker region endows human BK channels with peroxidase activity, showing an apparent H₂O₂ affinity approximately 40-fold greater than that of mitochondrial cytochrome c under baseline conditions. This peroxidase activity was reduced when substitutions were made at ⁶¹²CKACH⁶¹⁶ and other relevant sites. These results indicate that the BK channel possesses a novel module similar to the cytochrome c domains of hemoproteins, which may give rise to unique physiological functions for these widespread ion channels. Full article

The cellulose-binding domain and inter-domain linker play crucial roles in the degradation of crystalline cellulose by cellulases. Although significant differences exist in the degradation efficiency of cellobiohydrolase I (CBH I) derived from different fungal sources, the relationship between this efficiency diversity and variations in the non-catalytic region remains poorly understood. In this study, we found significant differences in the length and amino acid composition of the linker region of CBH I derived from Sordariomycetes and Eurotiomycetes. By replacing the non-catalytic region of Penicillium oxalicum CBH I with the corresponding segment from Trichoderma reesei, the cellulose conversion efficiency of the extracellular enzyme system doubled under the same protein dosage, and the adsorption of CBH I onto cellulose was improved. While replacing only the cellulose-binding domain improved the degradation efficiency of the enzyme system, additional replacement of the linker region resulted in greater enhancement. Improved degradation efficiency due to non-catalytic region replacement was observed under various conditions, including higher cellulose substrate concentration, reduced cellulose crystallinity, use of pretreated straw as a substrate, and degradation at physiological temperature. These findings provide novel insights into the molecular mechanisms underlying crystalline cellulose degradation by filamentous fungi. Full article

The BAHD acyltransferase family plays a critical role in plant secondary metabolism by catalyzing acyl transfer reactions that are essential for synthesizing metabolites involved in environmental adaptation. However, systematic investigation of this superfamily in Brassica napus has not been reported. In this study, 158 BnaBAHD genes were identified by comprehensive analyses of evolutionary relationships, motif structures, chromosomal distribution, gene collinearity, and selection pressures, and these genes were phylogenetically classified into five clades harboring conserved catalytic domains (HXXXD and DFGWG). Transient overexpression combined with metabolomic profiling demonstrated that two homologous seed-specific Clade V members, BnaBAHD040 and BnaBAHD120, which exhibited elevated expression during late seed development, significantly enhanced the accumulation of acylated metabolites contributing to biotic/abiotic stress resistance. This study provides the first experimental validation of the catalytic functions of BAHD enzymes in B. napus, establishing a theoretical foundation for leveraging this gene family in genetic improvement to develop novel rapeseed cultivars with enhanced stress tolerance and yield. Full article

Apigenin and sodium butyrate have been reported to help alleviate oxidative stress. This study evaluated the jejunal transcriptomic responses in ducks receiving apigenin and sodium butyrate supplementation under oxidative stress. In total, 200 healthy 300-day-old female Jinyun Ma ducks (1.53 kg ± 0.15) were randomly divided into four groups, with five replicates per group. The groups were as follows: a control group (CON): ducks were fed a basal diet with sterile saline injection; a diquat-injection (DIQ) group: ducks were fed a basal diet with diquat injection; an apigenin plus diquat group (API): ducks were fed a basal diet containing apigenin (500 mg/kg) with diquat injection; and a sodium butyrate plus diquat group (SB): ducks were fed a basal diet containing sodium butyrate (500 mg/kg) with diquat injection. The injection dose of diquat is 8 mg/kg body weight. Analysis revealed that the dietary supplementation of apigenin and sodium butyrate reduced malondialdehyde (MDA) levels and increased total antioxidant capacity (T-AOC) (p < 0.05). Compared to the DIQ group, sodium butyrate supplementation during oxidative stress elevated jejunal villus height and villus height/crypt depth ratio in ducks (p < 0.05). The study identified that some candidate genes, including solute carrier family 4 member 3 (SLC4A3), ADAM metallopeptidase domain 12 (ADAM12), and B-cell lymphoma 2-associated-athanogene 3 (BAG3), were significantly upregulated, whereas claudin 23 (CLDN23) and glucose-6-phosphatase catalytic subunit 1 (G6PC1) were markedly downregulated in the API group in comparison with that in the DIQ group (p < 0.05). Collectively, our findings provide molecular evidence for the beneficial effects of apigenin and sodium butyrate against oxidative stress in the jejunum of ducks. Full article

The BCR-ABL1 chimeric oncoprotein plays a pivotal role in the pathogenesis of Chronic Myeloid Leukemia (CML) as its constitutive kinase activity transforms the hematopoietic stem cell, promoting pro-survival signaling. We and others have previously shown that the manipulation of BCR-ABL1 catalytic activity modulates its intracellular localization, thereby transforming the culprit of CML into a pro-apoptotic protein that selectively kills leukemic cells. Here, we investigated the role of the BCR coiled-coil and C2 domains on BCR-ABL1 intracellular localization and leukemogenic potential. We performed a bioinformatic analysis that identified two putative nuclear localization signals (NLSs) in BCR. Using recombinant DNA strategies, we generated multiple BCR and BCR-ABL1 mutants that were ectopically expressed in human cells. The intracellular localization of each construct was analyzed by immunofluorescence, while their biological activity was investigated employing proliferation and transforming assays. We show that BCR displays two nuclear localization signals functionally inactivated by the coiled-coil and C2 domains. The removal of these regions reactivated the nuclear migration of both BCR and BCR-ABL1 mutants. Moreover, BCR-ABL1 constructs devoid of the coiled-coil and C2 domains displayed reduced transforming potential in Ba/F3 cells and in primary human CD34+ progenitors. Finally, we demonstrate that the deletion of the C2 domain compromises TKI efficacy. Our findings identify two nuclear localization signals in the BCR sequence that are functionally suppressed by the coiled-coil and C2 domains. Targeting these regions may provide additional therapeutic strategies to manipulate both BCR-ABL1 intracellular localization and kinase activity. Full article

Poly(ADP-ribosyl)ation is a crucial posttranslational modification that governs gene expression, chromatin remodeling, and cellular homeostasis. This dynamic process is mediated by the opposing activities of poly(ADP-ribose) polymerases (PARPs), which synthesize poly(ADP-ribose) (pADPr), and poly(ADP-ribose) glycohydrolase (PARG), which degrades it. While PARP function has been extensively studied, the structural and mechanistic basis of PARG-mediated pADPr degradation remain incompletely understood. To investigate the role of PARG in pADPr metabolism, we employed CRISPR/Cas9-based genome editing to generate a novel Parg^29b mutant mouse embryonic stem cell (ESC) line carrying a precise deletion within the PARG catalytic domain. This deletion completely abolished pADPr hydrolytic activity, resulting in massive nuclear pADPr accumulation, yet ESC viability, proliferation, and cell cycle progression remained unaffected. Using Drosophila melanogaster as a model system, we demonstrated that this mutation completely disrupted the pADPr pathway and halted developmental progression, highlighting the essential role of PARG and pADPr turnover in organismal development. Our results define a critical structural determinant of PARG catalytic function, underscore the distinct requirements for pADPr metabolism in cellular versus developmental contexts, and provide a genetically tractable model for studying the regulation of poly(ADP-ribose) dynamics and therapeutic responses to PARP inhibition in vivo. Full article

Nanozymes constitute a rapidly advancing frontier in scientific research, attracting widespread international interest, particularly for their role in facilitating cascade reactions. Despite their initial discovery a few years ago, significant hurdles persist in optimizing their catalytic performance and substrate specificity—challenges that are especially critical in the context of biomedical diagnostics. Within this domain, nitrogen-containing graphene oxide-based nanozymes exhibiting peroxidase-mimicking activity have emerged as particularly promising candidates, owing to the exceptional electrical conductivity, mechanical flexibility, and structural resilience of reduced graphene oxide-based materials. Intensive efforts have been devoted to engineering graphene oxide structures to enhance their peroxidase-like functionality. Nonetheless, the practical implementation of such nanozymes remains under active investigation and demands further refinement. This review synthesizes the current developments in nitrogen heteroatom-containing graphene oxide nanozymes and their derivative nanozymes, emphasizing recent breakthroughs and biomedical applications. It concludes by exploring prospective directions and the broader potential of these materials in the biomedical landscape. Full article

Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using microbial enzymes, especially cytochrome P450 monooxygenases (CYPs/P450s) and is well documented. Understanding the structural determinants that govern the regio- and stereoselectivity of steroid hydroxylation by P450s is essential in order to fully exploit their potential. Herein, we present a comprehensive analysis of the steroid-hydroxylating CYP109 family across the domains of life and delineate the structural determinants that govern steroid hydroxylation. Data mining, annotation, and phylogenetic analysis revealed that CYP109 family members are highly populated in bacteria, and indeed, these members passed from bacteria to archaea by horizontal gene transfer, leading to the evolution of P450s in archaea. Analysis of twelve CYP109 crystal structures revealed large, flexible, and dynamic active site cavities that can accommodate multiple ligands. The correct positioning and orientation of the steroid in the active site cavity and the nature of the C17 substituent on the steroid molecule influence catalysis. In an analogous fashion to the CYP107 family, the amino acid residues within the CYP109 binding pocket involve hydrophilic and hydrophobic interactions, influencing substrate orientations and anchoring and determining the site of hydroxylation and catalytic activity. A handful of amino acids, such as Val84, Val292, and Ser387 in CYP109B4, have been found to play a role in determining the catalytic regiospecificity, and a single amino acid, such as Arg74 in CYP109A2, has been found to be essential for the enzymatic activity. This work serves as a reference for the precise understanding of CYP109 structure–function relationships and for P450 enzymes in general. The findings will guide the genetic engineering of CYP109 enzymes to produce valuable steroid molecules of medicinal and biotechnological importance. Full article

Platinum-based catalysts remain the benchmark for the oxygen reduction reaction (ORR) in fuel cells, owing to their exceptional catalytic activity in the harsh chemical environment. However, optimizing Pt utilization and improving performance through support engineering are essential for commercial viability. In this study, we synthesized carbon-supported binary Pt-M (M = Ni, Cu, Co) electrocatalysts to investigate the influence of metal–support interactions on ORR activity. The Pt-M nanoparticles were fabricated on carbon supports, enabling the systematic screening of electronic and structural interactions. Among all compositions, Pt@Co exhibited the highest ORR mass activity, delivering 817 mA mg_Pt⁻¹ at 0.85 V and 464 mA mg_Pt⁻¹ at 0.90 V vs. RHE, surpassing both commercial Pt/C (J.M. 20 wt.%) and its Pt@Ni, Pt@Cu, and Pt@CNT counterparts. Structural and spectroscopic analyses reveal a strong electronic interaction between Pt and Co, leading to localized electron transfer from Co to Pt domains. This electronic modulation facilitates an optimal surface binding energy, enhancing oxygen adsorption–desorption kinetics and ORR activity. These findings highlight the critical role of transition metal–support synergy in the rational design of high-performance Pt-based electrocatalysts for next-generation fuel cell applications. Full article