Cancer-associated mutations in protein kinase C theta are loss-of-function.

The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.

Stabilization of insect cell membranes and soluble enzymes by accumulated cryoprotectants during freezing stress.

Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.

The crotonylated and succinylated proteins of jujube involved in phytoplasma-stress responses.

BACKGROUND: Protein posttranslational modifications (PTMs) are fast and early responses to environmental changes, including pathogen infection. Jujube witches' broom (JWB) is a phytoplasma disease causing great economic loss in jujube production. After phytoplasma infection, the transcriptional, translational, and metabolic levels in jujube were activated, enabling it to survive during phytoplasma invasion. However, no study has yet reported on PTMs in jujube. Lysine crotonylation (Kcr) and lysine succinylation (Ksu) have been popular studies in recent years and their function in plant phytoplasma-stress responses remains unclear. RESULTS: Here, 1656 crotonylated and 282 succinylated jujube proteins were first identified under phytoplasma-stress, of which 198 were simultaneously crotonylated and succinylated. Comparative analysis revealed that 656 proteins, 137 crotonylated and 43 succinylated proteins in jujube were regulated by phytoplasma infection, suggesting that Kcr was more universal than Ksu. Kcr differentially expressed proteins (DEPs) were related to ribosomes, photosynthetic and carbon metabolism, while Ksu DEPs were mainly involved in carbon metabolism, the TCA cycle and secondary metabolite biosynthesis. The crosstalk network among proteome, crotonylome and succinylome showed that DEPs related to ribosomal, peroxidases and glutathione redox were enriched. Among them, ZjPOD51 and ZjPHGPX2 significantly increased at the protein and Kcr level under phytoplasma-stress. Notably, 7 Kcr sites were identified in ZjPHGPX2, a unique antioxidant enzyme. After inhibitor nicotinamide (NAM) treatment, GPX enzyme activity in jujube seedlings was reduced. Further, site-directed mutagenesis of key Kcr modification sites K130 and/or K135 in ZjPHGPX2 significantly reduced its activity. CONCLUSIONS: This study firstly provided large-scale datasets of Kcr and Ksu in phytoplasma-infected jujube and revealed that Kcr modification in ZjPHGPX2 positively regulates its activity.

The role of N-terminal phosphorylation of DGK-θ.

Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To examine the role of phosphorylation on DGK-θ, we first identified the phosphorylated sites on endogenous DGK-θ from mouse brain and found four sites: S15, S17, which we refer to phosphomotif-1 sites, and S22 and S26 which we refer to as phosphomotif-2 sites. This study focused on the role of these phosphorylated sites on enzyme activity, membrane binding, thermal stability, and cellular half-life of DGK-θ. After generating a construct devoid of all non-catalytic phosphorylation sites (4A), we also generated other constructs to mimic phosphorylation of these residues by mutating them to glutamate (E). Our data demonstrate that an increase in membrane affinity requires the phosphorylation of all four endogenous sites as the phosphomimetic 4E but not other phosphomimietics. Furthermore, 4E also shows an increase in basal activity as well as an increase in the Syt1-induced activity compared to 4A. It is noteworthy that these phosphorylations had no effect on the thermal stability or cellular half-life of this enzyme. Interestingly, when only one phosphorylation domain (phosphomotif-1 or phosphomotif-2) contained phosphomimetics (S15E/S17E or S22E/S26E), the basal activity was also increased but membrane binding affinity was not increased. Furthermore, when only one residue in each domain mimicked an endogenous phosphorylated serine (S15E/S22E or S17E/S26E), the Syt1-induced activity as well as membrane binding affinity decreased relative to 4A. These results indicate that these endogenous phosphorylation sites contribute differentially to membrane binding and enzymatic activity.

Impact of Surfactants on Cumulative Trypsin Activity in Bottom-Up Proteome Analysis.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

Additive impact of soy protein dietary intake and exercise on visceral fat mass reduction and mitochondrial complex I activation in skeletal muscle.

Soy protein has shown remarkable effectiveness in reducing fat mass compared with other protein sources, and exercise has the potential to further enhance this fat loss effect. Previous studies have demonstrated that soy protein intake leads to decreased fatty acid synthesis, which contributes to its fat-loss properties. However, the exact mechanism by which these lipids are consumed remains unclear. To investigate this, we conducted a comprehensive study using C57/BL6 male mice, comparing the effects of soy and casein proteins with and without exercise (Casein-Sed, Casein-Ex, Soy-Sed, and Soy-Ex groups) under high- and low-protein conditions (14% or 40% protein). Our findings revealed that combining soy protein intake with exercise significantly reduced epididymal white adipose tissue (eWAT) weight, particularly in the high-protein diet group. Further analysis revealed that exercise increased the expression of lipid oxidation-regulatory proteins, including mitochondrial oxidative phosphorylation protein (OXPHOS) complexes, in the plantaris muscle regardless of the protein source. Although soy protein intake did not directly affect muscle mitochondrial protein expression, the activity of OXPHOS complex I was additively enhanced by exercise and soy protein under the 40% protein condition. Notably, complex I activity inversely correlated with eWAT weight in the soy protein diet group. These results highlight the potential link between improved complex I activity induced by soy protein and fat mass reduction, which emphasizes the promising benefits of combining soy protein with exercise in promoting fat loss.NEW & NOTEWORTHY The findings revealed that soy protein intake combined with exercise resulted in reduced adipose tissue weight compared with that obtained with casein protein intake. Furthermore, the joint impact of exercise and soy protein consumption resulted in enhanced activity of oxidative phosphorylation protein (OXPHOS) complex I in fast-twitch muscles, which appears to be associated with fat mass reduction. These findings elucidate the potential additive effects of soy protein and exercise on body weight management.

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer.

Phospholipases find versatile applications across industries, including detergent production, food modification, pharmaceuticals (especially in drug delivery systems), and cell signaling research. In this study, we present a strain of Bacillus paranthracis for the first time, demonstrating significant potential in the production of phosphatidylcholine-specific phospholipase C (PC-PLC). The investigation thoroughly examines the B. paranthracis PUMB_17 strain, focusing on the activity of PC-PLC and its purification process. Notably, the PUMB_17 strain displays extracellular PC-PLC production with high specific activity during the late exponential growth phase. To unravel the genetic makeup of PUMB_17, we employed nanopore-based whole-genome sequencing and subsequently conducted a detailed genome annotation. The genome comprises a solitary circular chromosome spanning 5,250,970 bp, featuring a guanine-cytosine ratio of 35.49. Additionally, two plasmids of sizes 64,250 bp and 5845 bp were identified. The annotation analysis reveals the presence of 5328 genes, encompassing 5186 protein-coding sequences, and 142 RNA genes, including 39 rRNAs, 103 tRNAs, and 5 ncRNAs. The aim of this study was to make a comprehensive genomic exploration that promises to enhance our understanding of the previously understudied and recently documented capabilities of Bacillus paranthracis and to shed light on a potential use of the strain in the industrial production of PC-PLC.

Succinimide Derivatives as Acetylcholinesterase Inhibitors-In Silico and In Vitro Studies.

We studied the effect of succinimide derivatives on acetylcholinesterase activity due to the interest in compounds that influence this enzyme's activity, which could help treat memory issues more effectively. The following parameters were established for this purpose based on kinetic investigations of the enzyme in the presence of succinimide derivatives: the half-maximal inhibitory concentration, the maximum rate, the inhibition constant, and the Michaelis-Menten constant. Furthermore, computational analyses were performed to determine the energy required for succinimide derivatives to dock with the enzyme's active site. The outcomes acquired in this manner demonstrated that all compounds inhibited acetylcholinesterase in a competitive manner. The values of the docking energy parameters corroborated the kinetic parameter values, which indicated discernible, albeit slight, variations in the inhibitory intensity among the various derivatives.

The correlation of asparaginase enzyme activity levels after PEG-asparaginase administration with clinical characteristics and adverse effects in Chinese paediatric patients with acute lymphoblastic leukaemia.

Studies on asparaginase enzyme activity (AEA) monitoring in Chinese patients receiving PEG-asparaginase remain limited. We monitored AEA in paediatric patients diagnosed with acute lymphoblastic leukaemia (ALL) and treated according to the Chinese Children's Cancer Group study protocols, CCCG-ALL-2015/CCCG-ALL-2020 protocols. We measured the AEA at days 7 ± 1 and 14 ± 1 and analysed their association with patient characteristics and PEG-asparaginase-related adverse effects (AEs). We measured 2147 samples from 329 patients. Mean AEA levels (interquartile range) were 931 iu/L (654-1174 iu/L) at day 7 ± 1 and 664 iu/L (463-860 iu/L) at day 14 ± 1. The AEA levels were higher in younger children and increased with the cumulative dose numbers. PEG-asparaginase inactivation rate was 19.1%, and the silent inactivation (SI) rate was 12.5%. Nine patients were identified with allergic-like reactions. Hypofibrinogenaemia, hypertriglyceridaemia, pancreatitis and thrombosis were associated with older age, whereas hypoglycaemia was associated with younger age. The risk of hypertriglyceridaemia and hypoglycaemia increased with cumulative dose numbers of PEG-asparaginase. Except for hypofibrinogenaemia, elevated AEA levels did not increase the risk of PEG-asparaginase-related AEs. Drug monitoring can be utilized as guidance for treatment decision-making. Individualizing asparaginase doses do not reduce toxicities. The treatment target of PEG-asparaginase remains to achieve sustained and adequate activity.

Transcriptome profiling reveals the impact of various levels of biochar application on the growth of flue-cured tobacco plants.

BACKGROUND: Biochar, a carbon-rich source and natural growth stimulant, is usually produced by the pyrolysis of agricultural biomass. It is widely used to enhance plant growth, enzyme activity, and crop productivity. However, there are no conclusive studies on how different levels of biochar application influence these systems. METHODS AND RESULTS: The present study elucidated the dose-dependent effects of biochar application on the physiological performance, enzyme activity, and dry matter accumulation of tobacco plants via field experiments. In addition, transcriptome analysis was performed on 60-day-old (early growth stage) and 100-day-old (late growth stage) tobacco leaves to determine the changes in transcript levels at the molecular level under various biochar application levels (0, 600, and 1800 kg/ha). The results demonstrated that optimum biochar application enhances plant growth, regulates enzymatic activity, and promotes biomass accumulation in tobacco plants, while higher biochar doses had adverse effects. Furthermore, transcriptome analysis revealed a total of 6561 differentially expressed genes (DEGs) that were up- or down-regulated in the groupwise comparison under different treatments. KEGG pathways analysis demonstrated that carbon fixation in photosynthetic organisms (ko00710), photosynthesis (ko00195), and starch and sucrose metabolism (ko00500) pathways were significantly up-regulated under the optimal biochar dosage (600 kg/ha) and down-regulated under the higher biochar dosage (1800 kg/ha). CONCLUSION: Collectively, these results indicate that biochar application at an optimal rate (600 kg/ha) could positively affect photosynthesis and carbon fixation, which in turn increased the synthesis and accumulation of sucrose and starch, thus promoting the growth and dry matter accumulation of tobacco plants. However, a higher biochar dosage (1800 kg/ha) disturbs the crucial source-sink balance of organic compounds and inhibits the growth of tobacco plants.

Nano silica's role in regulating heavy metal uptake in Calendula officinalis.

BACKGROUND: Soil contamination with heavy metals poses a significant threat to plant health and human well-being. This study explores the potential of nano silica as a solution for mitigating heavy metal uptake in Calendula officinalis. RESULTS: Greenhouse experiments demonstrated, 1000 mg•kg- 1 nano silica caused a 6% increase in soil pH compared to the control treatment. Also in 1000 mg. kg- 1 nano silica, the concentrations of available Pb (lead), Zn (zinc), Cu (copper), Ni (nickel), and Cr (chromium) in soil decreased by 12%, 11%, 11.6%, 10%, and 9.5%, respectively, compared to the control. Nano silica application significantly reduces heavy metal accumulation in C. officinalis exposed to contaminated soil except Zn. In 1000 mg.kg- 1 nano silica shoots Zn 13.28% increased and roots Zn increased 13% compared to the control treatment. Applying nano silica leads to increase the amount of phosphorus (P) 25%, potassium (K) 26% uptake by plant, In 1000 mg.kg - 1 treatment the highest amount of urease enzyme activity was 2.5%, dehydrogenase enzyme activity, 23.6% and the highest level of alkaline phosphatase enzyme activity was 13.5% higher than the control treatment. CONCLUSION: Nano silica, particularly at a concentration of 1000 mg.kg - 1, enhanced roots and shoots length, dry weight, and soil enzyme activity Moreover, it increased P and K concentrations in plant tissues while decreasing heavy metals uptake by plant.

The impact of root systems and their exudates in different tree species on soil properties and microorganisms in a temperate forest ecosystem.

BACKGROUND: The species composition of tree stands plays an important role in shaping the properties of forest soils. The aim of our research was to determine the influence on soil properties of the root systems of six species of trees which form forest stands in the temperate climatic zone. The research covered areas including six tree species - Scots pine (Pinus sylvestris L.), European larch (Larix deciduas Mill.), English oak (Quercus robur L.), English ash (Fraxinus excelsior L.), European beech (Fagus sylvatica L.) and European hornbeam (Carpinus betulus L.). In our study, we determined the characteristics of the roots and the amount of carbon excreted alongside their exudates. Enzymatic activity, and the composition and diversity of the fungi and bacteria, were also determined in addition to the basic physicochemical properties of the soil samples. RESULTS: A strong relationship between the root characteristics and soil properties, including the pH, basic cation content and phosphorus content, was confirmed. In addition, the enzymatic activity of phosphatase, ß-glucosidase, N-acetyl-ß-D-glucosaminidase and ß-D-cellobiosidase were positively correlated with the root characteristics. The study on soil bacteria across different tree species revealed Proteobacteria and Actinobacteriota to be the most abundant phylum. Fungal analysis showed Basidiomycota and Ascomycota as the dominant phyla. Ascomycota dominated in hornbeam and oak soils. Mortierellomycota was remarkably more present in pine soil. CONCLUSIONS: This analysis of root systems and soil properties confirmed the distinctness of ash stands, which were also more abundant in various microorganisms. It was also found that soils affected by different tree species were characterised by varied fungal and bacterial composition. The ash had particularly beneficial impact on soil microbiota.

Multi-Enzyme Activity of MIL-101 (Fe)-Derived Cascade Nano-Enzymes for Antitumor and Antimicrobial Therapy.

The clinical application of oncology therapy is hampered by high glutathione concentrations, hypoxia, and inefficient activation of cell death mechanisms in cancer cells. In this study, Fe and Mo bimetallic sulfide nanomaterial (FeS2@MoS2) based on metal-organic framework structure is rationally prepared with peroxidase (POD)-, catalase (CAT)-, superoxide dismutase (SOD)-like activities and glutathione depletion ability, which can confer versatility for treating tumors and mending wounds. In the lesion area, FeS2@MoS2 with SOD-like activity can facilitate the transformation of superoxide anions (O2 -) to hydrogen peroxide (H2O2), and then the resulting H2O2 serves as a substrate for the Fenton reaction with FMS to produce highly toxic hydroxyl radicals (∙OH). Simultaneously, FeS2@MoS2 has an ability to deplete glutathione (GSH) and catalyze the decomposition of nicotinamide adenine dinucleotide phosphate (NADPH) to curb the regeneration of GSH from the source. Thus it can realize effective tumor elimination through synergistic apoptosis-ferroptosis strategy. Based on the alteration of the H2O2 system, free radical production, glutathione depletion and the alleviation of hypoxia in the tumor microenvironment, FeS2@MoS2 NPS can not only significantly inhibit tumors in vivo and in vitro, but also inhibit multidrug-resistant bacteria and hasten wound healing. It may open the door to the development of cascade nanoplatforms for effective tumor treatment and overcoming wound infection.

Rationally Designed Functionalization of Single-Walled Carbon Nanotubes for Real-Time Monitoring of Cholinesterase Activity and Inhibition in Plasma.

Enzymes play a pivotal role in regulating numerous bodily functions. Thus, there is a growing need for developing sensors enabling real-time monitoring of enzymatic activity and inhibition. The activity and inhibition of cholinesterase (CHE) enzymes in blood plasma are fluorometrically monitored using near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) as probes, strategically functionalized with myristoylcholine (MC)- the substrate of CHE. A significant decrease in the fluorescence intensity of MC-suspended SWCNTs upon interaction with CHE is observed, attributed to the hydrolysis of the MC corona phase of the SWCNTs by CHE. Complementary measurements for quantifying choline, the product of MC hydrolysis, reveal a correlation between the fluorescence intensity decrease and the amount of released choline, rendering the SWCNTs optical sensors with real-time feedback in the NIR biologically transparent spectral range. Moreover, when synthetic and naturally abundant inhibitors inhibit the CHE enzymes present in blood plasma, no significant modulations of the MC-SWCNT fluorescence are observed, allowing effective detection of CHE inhibition. The rationally designed SWCNT sensors platform for monitoring of enzymatic activity and inhibition in clinically relevant samples is envisioned to not only advance the field of clinical diagnostics but also deepen further understanding of enzyme-related processes in complex biological fluids.

Determination of soil phenanthrene degradation through a fungal-bacterial consortium.

Fungal-bacterial consortia enhance organic pollutant removal, but the underlying mechanisms are unclear. We used stable isotope probing (SIP) to explore the mechanism of bioaugmentation involved in polycyclic aromatic hydrocarbon (PAH) biodegradation in petroleum-contaminated soil by introducing the indigenous fungal strain Aspergillus sp. LJD-29 and the bacterial strain Pseudomonas XH-1. While each strain alone increased phenanthrene (PHE) degradation, the simultaneous addition of both strains showed no significant enhancement compared to treatment with XH-1 alone. Nonetheless, the assimilation effect of microorganisms on PHE was significantly enhanced. SIP revealed a role of XH-1 in PHE degradation, while the absence of LJD-29 in 13C-DNA indicated a supporting role. The correlations between fungal abundance, degradation efficiency, and soil extracellular enzyme activity indicated that LJD-29, while not directly involved in PHE assimilation, played a crucial role in the breakdown of PHE through extracellular enzymes, facilitating the assimilation of metabolites by bacteria. This observation was substantiated by the results of metabolite analysis. Furthermore, the combination of fungus and bacterium significantly influenced the diversity of PHE degraders. Taken together, this study highlighted the synergistic effects of fungi and bacteria in PAH degradation, revealed a new fungal-bacterial bioaugmentation mechanism and diversity of PAH-degrading microorganisms, and provided insights for in situ bioremediation of PAH-contaminated soil.IMPORTANCEThis study was performed to explore the mechanism of bioaugmentation by a fungal-bacterial consortium for phenanthrene (PHE) degradation in petroleum-contaminated soil. Using the indigenous fungal strain Aspergillus sp. LJD-29 and bacterial strain Pseudomonas XH-1, we performed stable isotope probing (SIP) to trace active PHE-degrading microorganisms. While inoculation of either organism alone significantly enhanced PHE degradation, the simultaneous addition of both strains revealed complex interactions. The efficiency plateaued, highlighting the nuanced microbial interactions. SIP identified XH-1 as the primary contributor to in situ PHE degradation, in contrast to the limited role of LJD-29. Correlations between fungal abundance, degradation efficiency, and extracellular enzyme activity underscored the pivotal role of LJD-29 in enzymatically facilitating PHE breakdown and enriching bacterial assimilation. Metabolite analysis validated this synergy, unveiling distinct biodegradation mechanisms. Furthermore, this fungal-bacterial alliance significantly impacted PHE-degrading microorganism diversity. These findings advance our understanding of fungal-bacterial bioaugmentation and microorganism diversity in polycyclic aromatic hydrocarbon (PAH) degradation as well as providing insights for theoretical guidance in the in situ bioremediation of PAH-contaminated soil.

Arthropod prey type drives decomposition rates and microbial community processes.

Microbial communities perform various functions, many of which contribute to ecosystem-level nutrient cycling via decomposition. Factors influencing leaf detrital decomposition are well understood in terrestrial and aquatic ecosystems, but much less is known about arthropod detrital inputs. Here, we sought to infer how differences in arthropod detritus affect microbial-driven decomposition and community function in a carnivorous pitcher plant, Sarracenia purpurea. Using sterile mesh bags filled with different types of sterile arthropod prey, we assessed if prey type influenced the rate of decomposition in pitcher plants over 7 weeks. Additionally, we measured microbial community composition and function, including hydrolytic enzyme activity and carbon substrate use. When comparing decomposition rates, we found that ant and beetle prey with higher exoskeleton content lost less mass compared with fly prey. We observed the highest protease activity in the fly treatment, which had the lowest exoskeleton content. Additionally, we saw differences in the pH of the pitcher fluid, driven by the ant treatment which had the lowest pH. According to our results from 16S rRNA gene metabarcoding, prey treatments with the highest bacterial amplicon sequence variant (ASV) richness (ant and beetle) were associated with prey that lost a lower proportion of mass over the 7 weeks. Overall, arthropod detritus provides unique nutrient sources to decomposer communities, with different prey influencing microbial hydrolytic enzyme activity and composition. IMPORTANCE: Microbial communities play pivotal roles in nutrient cycling via decomposition and nutrient transformation; however, it is often unclear how different substrates influence microbial activity and community composition. Our study highlights how different types of insects influence decomposition and, in turn, microbial composition and function. We use the aquatic pools found in a carnivorous pitcher plant as small, discrete ecosystems that we can manipulate and study independently. We find that some insect prey (flies) breaks down faster than others (beetles or ants) likely because flies contain more things that are easy for microbes to eat and derive essential nutrients from. This is also reflected in higher enzyme activity in the microbes decomposing the flies. Our work bridges a knowledge gap about how different substrates affect microbial decomposition, contributing to the broader understanding of ecosystem function in a nutrient cycling context.

Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

The use of matrix-assisted laser desorption/ionization mass spectrometry in enzyme activity assays and its position in the context of other available methods.

Activity assays are indispensable for studying biochemical properties of enzymes. The purposes of measuring activity are wide ranging from a simple detection of the presence of an enzyme to kinetic experiments evaluating the substrate specificity, reaction mechanisms, and susceptibility to inhibitors. Common activity assay methods include spectroscopy, electrochemical sensors, or liquid chromatography coupled with various detection techniques. This review focuses on the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a growing and modern alternative, which offers high speed of analysis, sensitivity, versatility, possibility of automation, and cost-effectiveness. It may reveal reaction intermediates, side products or measure more enzymes at once. The addition of an internal standard or calculating the ratios of the substrate and product peak intensities and areas overcome the inherent inhomogeneous distribution of analyte and matrix in the sample spot, which otherwise results in a poor reproducibility. Examples of the application of MALDI-TOF MS for assaying hydrolases (including peptidases and ß-lactamases for antibiotic resistance tests) and other enzymes are provided. Concluding remarks summarize advantages and challenges coming from the present experience, and draw future perspectives such as a screening of large libraries of chemical compounds for their substrate or inhibitory properties towards enzymes.

Co(II) Substitution Enhances the Esterase Activity of a de Novo Designed Zn(II) Carbonic Anhydrase.

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH-)]N(TRIL2WL23H)3 n+ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH-)]N(TRIL2WL23H)3 + peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

A novel method for synthesizing authentic SARS-CoV-2 main protease.

The SARS-CoV-2 main protease (Mpro) plays a crucial role in virus amplification and is an ideal target for antiviral drugs. Currently, authentic Mpro is prepared through two rounds of proteolytic cleavage. In this method, Mpro carries a self-cleavage site at the N-terminus and a protease cleavage site followed by an affinity tag at the C-terminus. This article proposes a novel method for producing authentic Mpro through single digestion. Mpro was constructed by fusing a His tag containing TEV protease cleavage sites at the N-terminus. The expressed recombinant protein was digested by TEV protease, and the generated protein had a decreased molecular weight and significantly increased activity, which was consistent with that of authentic Mpro generated by the previous method. These findings indicated that authentic Mpro was successfully obtained. Moreover, the substrate specificity of Mpro was investigated. Mpro had a strong preference for Phe at position the P2, which suggested that the S2 subsite was an outstanding target for designing inhibitors. This article also provides a reference for the preparation of Mpro for sudden coronavirus infection in the future.