Search Results (2,568)

Acetolactate synthase (ALS; EC 2.2.1.6) catalyzes the first committed step in branched-chain amino acid (BCAA) biosynthesis and is the molecular target of multiple herbicide classes, including the imidazolinones. Here, we performed a genome-wide characterization of the HaALS gene family in sunflower (Helianthus annuus L.) and investigated genotype-dependent transcriptional responses to imazethapyr. A total of 11 HaALS genes were identified and classified into three phylogenetic clades (Groups A–C). All HaALS proteins contained the conserved TPP_enzyme domains (TPP_enzyme_N, TPP_enzyme_M, and TPP_enzyme_C), and purifying selection (Ka/Ks < 1) indicated strong evolutionary constraint on their core enzymatic function. Promoter analyses revealed abundant cis-regulatory elements associated with diverse stress and signaling inputs, supporting regulatory potential for herbicide-triggered transcriptional modulation. qRT-PCR analysis following imazethapyr application (0, 24, and 48 h) showed pronounced genotype-dependent expression reprogramming between the susceptible (S) and resistant (R) cultivars. In the R genotype, multiple HaALS members were strongly induced after treatment; specifically, HaALS4 reached a ~6-fold increase at 24 h and a >10-fold increase at 48 h, and HaALS11 increased to ~6–7-fold at 24 h while remaining above the baseline at 48 h; several additional paralogs exhibited intermediate induction (~3–8-fold by 48 h). In contrast, the S genotype showed limited changes (typically ~0.8–2-fold). Collectively, these findings define the evolutionary features of the sunflower HaALS family and identify herbicide-responsive paralogs that may contribute to imidazolinone tolerance, providing candidates for functional validation and molecular breeding. Full article

Artisanal raw-milk cheese represents a complex biochemical ecosystem where the indigenous microbiota acts as the primary driver of the volatile profile. This study utilizes an innovative synchronized biological relay model to decipher the mechanistic interplay between the successional dynamics of indigenous lactic acid bacteria (LAB) and the temporal evolution of the volatile metabolome over a 10-week maturation period of an artisanal cow-milk cheese. Utilizing a culture-dependent approach focused on the quantitative enumeration of broad morpho-physiological groups—without species-level identification—integrated with HS-SPME/GC-MS, we characterized the precise shifts from early-stage lactic cocci to dominant rod-shaped lactobacilli. Initial populations at Week 0 consisted of 8.2 log CFU/g of cocci and 4.1 log CFU/g of rod-shaped LAB. Lactic cocci peaked at Week 2 (8.5 log CFU/g) before undergoing mass autolysis down to 7.1 log CFU/g by Week 4, releasing intracellular enzymes that catalyzed a 900% surge in total esters and a 215% increase in volatile alcohols. Concurrently, rod-shaped LAB proliferated to a maximum of 8.6 log CFU/g at Week 6, directly correlating with a 125% increase in total carboxylic acids, prominently driven by a 750% accumulation of hexanoic acid. The late-phase maturation (Weeks 8–10) established a technological equilibrium: volatile sulfur compounds collapsed by over 90% within the first two weeks, initial transient lactones were replaced by a 1200% late-stage increase in dodecalactone, and matrix-sequestered dietary terpenes were liberated via an 8-fold (700%) increase at Week 8. This study introduces an innovative, statistically validated volatilomic framework that equips the dairy sector with an advanced metabolomic tool for rigorous product authentication and targeted flavor optimization, thereby establishing a scientific baseline for the reproducible production of premium, organoleptically superior artisanal cheeses. Full article

Nicotine is the main psychoactive component of tobacco and is considered to be the main substance responsible for the development of tobacco addiction. The main enzyme responsible for nicotine metabolism, CYP2A6, catalyzes the conversion of nicotine to cotinine and the subsequent metabolism of cotinine to trans-3′-hydroxycotinine. CYP2A6 activity is known to be modulated by various compounds, such as quercetin. This repeated-measures study examined the effects of short-term quercetin supplementation on urinary nicotine metabolism biomarkers in adult users of conventional and alternative nicotine products. Seventy-two participants completed a two-week study protocol involving first-morning urine collection at four time points: baseline, immediately after three days of quercetin supplementation (500 mg/day), seven days after supplementation, and ten days after supplementation. Urinary nicotine, cotinine, and trans-3′-hydroxycotinine concentrations were measured, and the nicotine metabolite ratio was calculated as trans-3′-hydroxycotinine/cotinine. Repeated-measures analysis of variance was used to evaluate biomarker changes over time according to sex, nicotine product type, and self-reported nicotine consumption intensity. Quercetin supplementation did not consistently alter nicotine metabolism biomarkers, while a descriptive increase in median urinary nicotine concentration after supplementation was observed in participants reporting lower daily nicotine consumption compared with other groups. These findings suggest that further studies are warranted to better clarify the effects of quercetin on nicotine metabolism across different levels of nicotine exposure. Full article

The hormonal vitamin D metabolite, 1,25-dihydroxyvitamin D (1,25D), produced primarily in the kidney, acts in numerous end-organs via the nuclear vitamin D receptor (VDR) to trigger molecular events that orchestrate bone mineral homeostasis, immune responsiveness, and aspects of behavior. Tyrosine hydroxylase (TH) encodes a neuronally expressed enzyme that catalyzes the initial, rate-limiting step in the production of several catecholamine neurotransmitters and hormones, including dopamine, norepinephrine, and epinephrine. Herein we report that TH mRNA is significantly induced (2.5-fold) and NURR1 mRNA is induced 9.3-fold by 10 nM 1,25D in differentiated human SH-SY5Y neuroblastoma cells. Similar results were observed in human U87 glioblastoma cells (TH, 2.6-fold; NURR1, 3.6-fold). Comparative analysis of TH gene promoter-proximal sequences from human, mouse, and rat identifies candidate NURR1-responsive elements (NBREs) at the following positions: −35, −855, −1470, and −2343 bp in the human gene; −34 and −961 bp in the mouse gene; and −34, −350, and −873 bp in the rat gene, consistent with NURR1 acting as a recurring regulatory factor at TH promoters across mammalian species. Furthermore, by interrogating VDR ChIP-seq/cistrome datasets, we identified candidate vitamin D-responsive elements (VDREs) at the human NURR1 locus that provide a plausible genomic framework for direct regulation of NURR1 by 1,25D/VDR. We propose that 1,25D-liganded VDR acts as a primary inducer of NURR1, which in turn secondarily activates expression of the TH gene, thereby defining a transcriptional route through which 1,25D/VDR signaling may influence TH-linked dopaminergic gene programs. Retinoid X receptor (RXR) may facilitate both NURR1-dependent and -independent potentiation of TH transcription because the rexinoid, bexarotene, significantly enhances TH mRNA in human U87 cells, either alone (2.0-fold) or in combination with 1,25D (4.1-fold). In addition, bexarotene and its novel analogs, A41 and A55, induce NURR1 mRNA expression in U87 cells by 2.8-, 3.1-, and 4.8-fold, respectively, with A55 outperforming the parent compound at matched concentration. Because Parkinson’s disease is characterized by the selective degeneration of dopaminergic neurons and impaired NURR1-dependent transcriptional programs, our findings identify a 1,25D/VDR–NURR1–RXR transcriptional axis as a previously underappreciated regulatory framework for studying TH gene expression and dopaminergic gene regulation in Parkinson’s disease-relevant neural contexts. Full article

Moringa oleifera is rich in γ-aminobutyric acid (GABA), a functional non-protein amino acid with significant antihypertensive and neuroprotective activities. However, the key enzymes responsible for catalyzing the conversion of L-glutamate (L-Glu) to GABA—glutamate decarboxylases (GADs)—have not been functionally characterized in M. oleifera, which limits its metabolic engineering applications. In this study, the previously obtained MoGAD1 (PZ458702) and MoGAD2 (PZ458703) genes were heterologously expressed in Escherichia coli Rosetta (DE3) to produce recombinant proteins. SDS-PAGE and Western blot analyses showed that both MoGAD1 and MoGAD2 were solubly expressed at 20 °C and 37 °C. Their catalytic functions were verified via whole-cell biocatalysis, and high-performance liquid chromatography (HPLC) analysis confirmed that both MoGAD1 and MoGAD2 could convert L-Glu to GABA. The GABA yields of the engineered strains harboring MoGAD1 and MoGAD2 reached 3.67 ± 0.1833 g/L and 0.648 ± 0.002 g/L, with conversion rates of 61.2% and 10.8%, respectively. Both MoGAD1 and MoGAD2 exhibited favorable docking with PLP, with binding energies of −5.489 kcal/mol and −5.297 kcal/mol, respectively; they also showed good docking with L-Glu, with binding energies of −4.207 kcal/mol and −4.49 kcal/mol, respectively. This study provides the first experimental evidence for the activity of the MoGAD protein encoded by the GAD gene from M. oleifera, elucidates the molecular mechanism underlying GABA accumulation, and offers candidate genes for biotechnological production of GABA. Full article

Furanocoumarins and their derivatives are found in various plant species and have attracted considerable attention due to their diverse biological activities. By analyzing the genomes of Apium Graveolens L. and Peucedanum praeruptorum Dunn, we characterized a set of candidate genes encoding key enzymes involved in furanocoumarin biosynthesis, including one prenyltransferase (AgPT1), cyclases (AgCOC1, PpCOC1 and PpCOC2), and methyltransferases (AgOMT1 and AgOMT2). Functional validation in Saccharomyces cerevisiae demonstrated that AgCOC1 and PpCOC2 accept both linear and angular substrates, whereas PpCOC1 accepts only linear substrates. Depending on the reaction conditions, these cyclases can produce compounds with either furan or pyran scaffolds. These findings reveal a previously unappreciated catalytic versatility of cyclases involved in furanocoumarin biosynthesis. Notably, the genes encoding the prenyltransferase and cyclases were found to be co-localized in the genome, which may significantly enhance the efficiency of furanocoumarin biosynthesis. This mechanism may account for the pronounced accumulation of furanocoumarins in Apiaceae plants. Finally, we provide the first evidence that AgOMT1 functions as a multifunctional methyltransferase capable of catalyzing the O-methylation modifications observed in furanocoumarins in A. graveolens. In conclusion, this study fills a research gap in our understanding of furanocoumarin biosynthesis and reveals that genes encoding cyclases and prenyltransferases are clustered in the genome, a pattern that arose during evolution. Full article

Nanozymes, nanomaterials that mimic enzymatic activity, offer superior stability, tunability, and lower production costs compared to natural enzymes. To date, most nanozyme-based point-of-care (PoC) diagnostic systems have relied on oxidation reactions, such as oxidation of 3,3′,5,5′-tetramethylbenzidine, which often suffer from limited substrate stability and high background signal. This study investigates reduction reactions, particularly those involving tetrazolium salts, as an alternative route for signal generation in PoC devices. For this purpose, monometallic and bimetallic gold, palladium, and platinum nanoparticles were synthesized via chemical reduction using poly(vinyl alcohol) as a stabilizing agent. The resulting nanoparticles were uniform in size and morphology. Their catalytic performance was confirmed through the reduction of 4-nitrophenol. The tetrazole salts were selected as promising substrates for application in PoC settings and further explored by examining the nanozyme-based reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). The nanozymes catalyzed the reduction of MTT in the presence of sodium borohydride, producing a distinct colorimetric signal under selected conditions. The effects of reducing agent concentration, buffer pH, and potential interferents were evaluated, with performance suitable for PoC devices achieved at basic pH and low borohydride concentration. Interference studies showed negligible MTT reduction in the presence of physiological levels of ascorbic acid, human serum albumin, and 10% concentration of human serum. Finally, a proof-of-concept lateral flow assay demonstrated successful signal generation through nanozyme-catalyzed MTT reduction. Results establish tetrazolium salts as suitable substrates for nanozyme-enhanced PoC diagnostics and highlight reduction-based chromogenic systems as a viable alternative to traditional oxidation-based assays. Full article

Chlorophyll (Chl) b is crucial for assembly of the light-harvesting antennae that are required for optimal photosynthetic activity in plants and green algae. Synthesis of its precursor, chlorophyllide (Chlide) b, is catalyzed by Chlide a oxygenase (CAO), which contains a stable tyrosyl radical. Studies with the model organism Chlamydomonas reinhardtii y-1 suggested that protochlorophyllide (Pchlide) a is a substrate for the enzyme in the dark when a ‘cofactor’ is present to form a heterodimer, which apparently decreases the redox potential of Pchlide a. Data described in the literature are consistent with reduction in the redox potential of Chlide a by dimerization, which produces a substrate that allows rapid synthesis and accumulation of Chl b during chloroplast development in oxygenic photosynthetic organisms. In this article, we provide an emerging perspective on CAO’s structure, its assumed radical-mediated catalytic mechanism, and its role in planta. Full article

Background/Objectives: Aromatic L-amino acid decarboxylase deficiency (AADC-D; OMIM #608643) is a rare autosomal recessive neurometabolic disorder caused by pathogenic variants in the DDC gene, leading to impaired of monoamine neurotransmitter biosynthesis. AADC, a pyridoxal-5′-phosphate (PLP)-dependent enzyme, catalyzes the conversion of L-dopa and 5-hydroxytryptophan (5-HTP) to dopamine and serotonin, respectively. Early diagnosis remains challenging due to the limited specificity of current biochemical approaches. This study aimed to evaluate plasma AADC enzyme activity using these physiological substrates by High-Performance Liquid Chromatography (HPLC)-based method and assess its potential utility in the biochemical diagnosis of AADC deficiency. Methods: Plasma AADC activity was quantified using physiological substrates (L-dopa and 5-HTP) by HPLC with electrochemical and fluorescence detection. Sanger sequencing of the DDC gene was performed in two suspected patients to identify pathogenic variants. Results: Two genetically confirmed AADC-D patients demonstrated reduced enzyme activity. Using L-dopa as substrate, enzyme activity in patients was 12.4 and 26.1 pmol/min/mL, both below the published reference interval (36–129 pmol/min/mL). Using 5-HTP as substrate, enzyme activity was 1.5 and 5.1 pmol/min/mL; Patient 1 showed activity below the reference interval (2.0–7.1 pmol/min/mL), while Patient 2 demonstrated activity within the lower range of reported values. Reduced enzyme activity was consistent with the clinical features and molecular findings with identification of pathogenic variants in the DDC gene (c.175G>A and c.714+4A>T). Conclusions: Plasma AADC activity measurement demonstrates potential as a functional biochemical biomarker that augments molecular genetic testing in the biochemical evaluation of AADC deficiency. Further studies involving larger patient cohorts are required to further evaluate its diagnostic performance and broader clinical applicability. Full article

β-Galactosidase is an important enzyme for lactose hydrolysis because it catalyzes the conversion of lactose into glucose and galactose. In this study, Aspergillus niger C18, which showed β-galactosidase-producing ability during preliminary screening, was selected as the gene source. A β-galactosidase gene from this strain was cloned into the pET28a vector and heterologously expressed in Escherichia coli. Solid-state fermentation conditions were optimized to produce the native enzyme as a reference for comparison. The enzymatic properties of the recombinant enzyme were then systematically characterized and compared with those of the native enzyme. The recombinant β-galactosidase exhibited favorable thermal and pH stability. After incubation for 2 h at its optimal pH and optimal temperature, the recombinant enzyme retained 88.9% and 94.1% of its initial activity, respectively; specifically, 88.9% corresponded to pH stability and 94.1% corresponded to thermal stability. These results indicate favorable stability of the recombinant enzyme under the tested conditions. Thin-layer chromatography and high-performance liquid chromatography analyses confirmed that the recombinant enzyme efficiently hydrolyzed lactose in a model lactose solution, achieving more than 99.0% lactose degradation after 12 h of reaction. These findings suggest that β-galactosidase derived from A. niger C18 is a promising candidate for lactose hydrolysis. Full article

RelA/SpoT homologue family enzymes participate in controlling the cellular levels of the alarmone (p)ppGpp, thereby activating the stringent response and promoting survival under stress conditions. These proteins contain an N-terminal catalytic domain and a C-terminal regulatory domain. They catalyze both the synthesis of ppGpp/pppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate. Here, we report the crystal structure of the N-terminal domain of Rel from Streptococcus equisimilis in complex with pppGpp at 3.2 Å resolution. The asymmetric unit contains a dimer with asymmetric ligation: pppGpp occupies only the synthetase site in one monomer, whereas in the other monomer, it is bound in both the hydrolase and synthetase sites. The two monomers exhibit distinct conformational states, with pronounced rearrangements of the flexible loops surrounding the binding pockets, including the α2/α3 and α8/α9 loops that act as steric gates. Molecular dynamics simulations support the dual binding arrangement and reveal additional probable transient binding sites, including a region in the linker between hydrolase and synthetase subdomains. These findings provide a structural framework for understanding how pppGpp binding modulates the opposing catalytic activities of bifunctional Rel enzymes and suggest possible mechanisms for (p)ppGpp-mediated autoregulation. Full article

Background: Uridine diphosphate glucose (UDP-Glc) is one of the key substrates for the biosynthesis of gallotannins in plants. UDP-glucose dehydrogenase (UGD) catalyzes the irreversible oxidation of UDP-Glc to UDP-glucuronic acid (UDP-GlcA), thus affecting the biosynthesis and accumulation of gallotannins in the Chinese gallnut. Methods and Results: In this study, we identified three members of the RcUGD family from the Rhus chinensis genome. Protein sequence alignment revealed that all three RcUGDs possess the conserved NAD⁺ coenzyme binding motif GAGYVGG and the catalytic motif GFGGSCFQKDIL. qRT-PCR analysis revealed that the expression levels of RcUGD3 in stem and root tissues were respectively 10-fold and 13-fold greater than that in the leaves, in which gallotannin accumulation was higher. RcUGD3 expression level declined by 63% during early (24 d) gallnut development, suggesting an inverse relationship between RcUGD3 expression level and gallotannin biosynthesis. In addition, subcellular localization analysis using the tobacco transient transformation system showed that RcUGD proteins are broadly distributed throughout the cell. Moreover, an in vitro enzyme activity assay indicated that the recombinant RcUGD3 protein catalyzed UDP-Glc to produce UDP-GlcA as shown by HPLC. Taken together, our results suggested that RcUGD3 protein is responsible for UDP-Glc degradation and probably plays a regulatory role in gallotannin biosynthesis in the Chinese gallnut. Conclusions: This study lays a foundation for further elucidating the function and expression regulation mechanism of the RcUGD gene family and provides new insights for the super-accumulation mechanisms of gallotannins in Chinese gallnuts. Full article

Human urine is a sustainable source of nutrients with significant fertilizer potential. The presence of pharmaceutical residues, however, obstructs its use in agriculture. Also, the loss of ammonia formed in enzyme-catalyzed hydrolysis of urea in stored urine compromises the approach. This study presents gas-phase pulsed corona discharge (PCD) used in the oxidation of pharmaceuticals and enzymes, improving the applicability of urine as a fertilizer. The prioritized beta-blocker propranolol (PR) and antibiotic tetracycline were chosen as target micropollutants for the experimental study, demonstrating enhanced oxidation relative to matrix constituents. Tetracycline showed its more recalcitrant character in urine than PR for its more pronounced matrix-mediated scavenging or complexation. The PCD oxidation significantly lowered the urease enzyme activity, thus preventing nitrogen loss through ammonia volatilization. According to the phytotoxicity assessment using the Pisum sativum garden peas test, urine PCD-treated with the energy dose sufficient to substantially degrade pharmaceuticals is not phytotoxic when applied at recommended agronomic doses. The findings reveal the use of PCD as an energy-efficient technology for producing safe and stable urine-derived fertilizer. Full article

Castration-resistant prostate cancer (CRPC) remains a significant clinical challenge due to the ability of tumor cells to undergo intratumoral androgen synthesis, a process catalyzed by the CYP17A1 enzyme. The only CYP17A1 inhibitor available in therapy, abiraterone acetate, faces significant limitations due to its steroidal structure, which causes off-target effects and generates agonistic metabolites that paradoxically stimulate the androgen receptor (AR). This study presents the development of the D2AAK1M series, a novel class of non-steroidal potential CYP17A1 inhibitors based on a pyridine–piperidine scaffold. Through biomimetic design and molecular docking, we demonstrated that these compounds have the potential to coordinate the heme iron while achieving high shape complementarity within the catalytic pocket. In silico ADME profiling indicated superior physicochemical properties compared to abiraterone, including optimal lipophilicity, enhanced water solubility, and the potential to penetrate the blood–brain barrier for targeting CNS metastases. In vitro assay results correlated with a suggested mechanism, showing preferential cytotoxicity toward androgen-dependent LNCaP cells (AR+) while sparing AR-negative lines (DU145, PC3) and healthy human fibroblasts (BJ). Our compounds present a promising starting point for further development of non-steroidal CYP17A1 inhibitors. Full article

This study examines the nature of enzymatic degradation of polyethylene terephthalate (PET) films mediated by a novel recombinant LCC^ICCG PETase enzyme preparation based on P. verruculosum fungus. The investigation was conducted using amorphous PET samples and PET samples with varying degrees of crystallinity as substrates for PETase-catalyzed hydrolysis under different temperature and pH conditions. Mechanical testing revealed that enzymatic treatment reduced the yield stress by 20–25%, tensile strength by approximately twofold, and elongation at break by 5–10 times, while the deformation mechanism remained unchanged. Enzymatic degradation under acidic conditions was ineffective, whereas increasing the pH to 9–10 markedly accelerated PET degradation and the associated deterioration of mechanical properties. Thermal analysis (TGA, DSC) and microscopy (optical and scanning electron microscopy) demonstrated that degradation was localized at the polymer surface, leading to the formation of cavities, cracks, and submicron-sized pores rather than bulk material disintegration. An inverse correlation was observed between PET crystallinity and susceptibility to enzymatic degradation: samples with crystallinity below 13% could be almost completely degraded, whereas samples with crystallinity above 30% exhibited little or no measurable weight loss over the same period. Low-crystallinity PET underwent rapid degradation accompanied by a transient increase in crystallinity, while highly crystalline PET primarily accumulated surface defects that nevertheless caused a substantial loss of mechanical strength. Consequently, the experimental data obtained in this study provide useful information for understanding PET degradation and for future studies on enzymatic PET recycling. The systematization of feedstock characteristics and the elucidated patterns of enzymatic degradation will enable optimization of pretreatment, enzymatic hydrolysis, and monomer recovery process parameters, thereby facilitating the eventual production of secondary raw materials. Full article