Isocitrate Lyase Activity Research Articles

Cheminformatics-enhanced discovery of therapeutic agents targeting isocitrate lyase in Mycobacterium tuberculosis infections

Tuberculosis (TB) is a global health challenge; therefore, there is an urgent requirement to develop a novel and more effective anti-TB therapeutic. This study targeted the isocitrate lyase (ICL) protein due to its pivotal role in the pathogenicity of Mycobacterium tuberculosis (Mtb). Virtual screening of 8752 bioactive compounds used an ML-based QSAR model and molecular docking. ADMET testing was performed on the top three hits to identify the compound most closely mimicking a drug molecule. The top hits, 648 and 2785758, showed high binding affinity towards ICL with −7.3 and −7 kcal/mol, comparable to the control. These molecules also showed strong binding with the residue Asp108, which plays a vital role in ICL activity. Molecular dynamics simulations showed stability for 648 and 2785758, comparable to the control compound used in this study. It was found that 648 bound to the protein maintained the RMSD constant and consistent at 0.3 nm for a complete 100 ns simulation. 2785758 showed a comparable RMSD trend to the control. Both 648 and 2785758 showed high RMSF for critical residue Asp108. Further, PCA and FEL confirmed the formation of a stable complex. MM/GBSA estimations of binding free energy indicated that compounds 648 had an elevated level of stability (ΔG TOTAL = −28.11 kcal/mol) and 2785758 (ΔG TOTAL = −21.05 kcal/mol). This study suggests that compounds 648 and 2785758 can potentially affect the activity of ICL, leading to its inactivation and ultimately preventing the progression of tuberculosis. Communicated by Ramaswamy H. Sarma

Conversion of lipids into carbohydrates rescues energy insufficiency in rapeseed germination under waterlogging stress.

Waterlogging stress, particularly during seed germination, significantly affects plant growth and development. However, the physiological and molecular mechanisms underlying waterlogging stress responses during rapeseed germination remain unclear. In this study, two rapeseed cultivars, Xiangzayou518 (waterlogging-sensitive) and Dadi199 (waterlogging-tolerant), were used to explore the physiological mechanisms underlying rapeseed response to waterlogging stress during germination. Our results showed that waterlogging significantly decreased the emergence percentage and seedling growth rate. During the radicle elongation period (from 48 to 96 h post-germination), the most sensitive period to waterlogging during germination, sugar content, and glycolysis efficiency were significantly decreased, but anaerobic fermentation was enhanced. In tolerant cultivars, when the energy supply was insufficient, the conversion efficiency of lipids into sugar increased, and the activities of isocitrate lyase, malate synthase, and fructose-1, 6-diphosphatase were enhanced by 11.63, 19.06, and 20.37%, respectively, at 72 h post-germination under waterlogging stress. Transcriptome data showed that the differentially expressed genes were significantly enriched in glucose and lipid metabolism pathways when comparing waterlogged stress and normal conditions. These results indicate that waterlogging affects seed germination in rapeseed by inhibiting carbohydrate metabolism, and the conversion capacity of lipids into sugar under waterlogging stress was stronger in tolerant cultivars than in sensitive cultivars, thus rescuing the insufficient energy supply in seed germination and seedling growth. This study reveals the physiological mechanism of rapeseed response to waterlogging stress during seed germination and provides a valuable reference for improving waterlogging tolerance.

The ICL1 and MLS1 Genes, Integral to the Glyoxylate Cycle, are Essential and Specific for Caloric Restriction-Mediated Extension of Lifespan in Budding Yeast.

The regulation of complex energy metabolism is intricately linked to cellular energy demands. Caloric restriction (CR) plays a pivotal role in modulating the expression of genes associated with key metabolic pathways, including glycolysis, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle. In this study, the chronological lifespan (CLS) of 35 viable single-gene deletion mutants under both non-restricted and CR conditions, focusing on genes related to these metabolic pathways is evaluated. CR is found to increase CLS predominantly in mutants associated with the glycolysis and TCA cycle. However, this beneficial effect of CR is not observed in mutants of the glyoxylate cycle, particularly those lacking genes for critical enzymes like isocitrate lyase 1 (icl1Δ) and malate synthase 1 (mls1Δ). This analysis revealed an increase in isocitrate lyase activity, a key enzyme of the glyoxylate cycle, under CR, unlike the activity of isocitrate dehydrogenase, which remains unchanged and is specific to the TCA cycle. Interestingly, rapamycin, a compound known for extending lifespan, does not increase the activity of the glyoxylate cycle enzyme. This suggests that CR affects lifespan through a distinct metabolic mechanism.

The second messenger (cyclic diguanylate and autoinducer-2) promotes N-acylated-homoserine lactones-based quorum sensing for enhanced recovery of stored aerobic granular sludge

Efficient quorum sensing (QS) response is the premise for recovering the activities of stored aerobic granular sludge (AGS). This study aims to explore the crosstalk between the secondary messenger and the N-acylated-homoserine lactones (AHLs) to yield protein-rich granules efficiently from stored AGS by enhancing its QS efficiency selectively. 80 nmol/L cyclic diguanylate (c-di-GMP) with 20 nmol/L AHLs could increase the activity of isocitrate lyase activity (ICD) by 89 % and isocitrate dehydrogenase activity (ICDHc) by 113.5 %, to accelerate the tricarboxylic acid (TCA) cycle for yielding excess proteins by 166.4 %. In contrast, 80 nmol/L autoinducer-2 (AI-2) with 20 nmol/L AHLs could increase the activities of ICD and ICDHc by 485 % and 54.5 %, respectively, accelerating the glyoxylate (GCA) cycle to activate fat acid synthesis for stimulating polysaccharides (PS) secretion by 137.9 %. The strategy with c-di-GMP successfully recovers the refrigerated-stored and dried-stored AGS into proteins-rich AGS, with enriched functional strains for the PN secretion.

The Transcription Factor StuA Regulates the Glyoxylate Cycle in the Dermatophyte Trichophyton rubrum under Carbon Starvation.

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.

Isolation and purification of isocitrate lyase by chromatographic meth-ods from the liver of rats under conditions of alloxan-induced diabetes

The homogeneous preparation with isocitrate lyase activity was obtained from the peroxisomal fraction of rat hepatocytes using chromatographic methods. Male white inbred laboratory Wistar rats (Rattus norvegicus) were used as the object of the study. Experimental diabetes was induced by a single injection of a 5% alloxan solution. The development of diabetes was monitored by measuring blood glucose levels using a glucometer (SatellitePlus PKG-02.4). Blood sampling was carried out in the morning from the tail vein. On the 10th day of the experiment, laboratory animals were subjected to decapitation, previously euthanized with ether anaesthesia, to obtain liver tissue samples. For the isolation of a homogeneous preparation of isocitrate lyase (ICL, EC 4.1.3.1.), chromatographic methods were used. Purification included several stages: fractionation of the homogenate with ammonium sulphate, gel filtration on columns filled with Sephadex G-25 and ion exchange chromatography, which was the determining stage. The anion exchanger DEAE-Sephacel was used as a sorbent. This allowed to achieve a degree of purification of 12.12 for the first ICL isoform and 24.24 for the second ICL isoform. The specific activity for the first sample was 0.04 U/mg protein, and the yield was 26.3%. The specific activity value for the second form of ICL was 0.08 U/mg protein, and the yield was 47.4%. For elution from a column filled with DEAE-Sephacel was performed using step KCl gradient (60 mM – 200 mM). Identification of the resulting preparations was carried out spectrophotometrically, following the increase in the optical density, according to the Kornberg method (λ-324 nm). The presence of isoforms was determined by specific staining of the gel after PAGE electrophoresis. As a result of four-step purification, two isoforms with different from ICL (EC 4.1.3.1) from other sources electrophoretic mobility were obtained.

Itaconic acid and dimethyl itaconate exert antibacterial activity in carbon-enriched environments through the TCA cycle

Itaconic acid (IA), a metabolite generated by the tricarboxylic acid (TCA) cycle in eukaryotic immune cells, and its derivative dimethyl itaconate (DI) exert antibacterial functions in intracellular environments. Previous studies suggested that IA and DI only inhibit bacterial growth in carbon-limited environments; however, whether IA and DI maintain antibacterial activity in carbon-enriched environments remains unknown. Here, IA and DI inhibited the bacteria with minimum inhibitory concentrations of 24.02 mM and 39.52 mM, respectively, in a carbon-enriched environment. The reduced bacterial pathogenicity was reflected in cell membrane integrity, motility, biofilm formation, AI-2/luxS, and virulence. Mechanistically, succinate dehydrogenase (SDH) activity and fumaric acid levels decreased in the IA and DI treatments, while isocitrate lyase (ICL) activity was upregulated. Inhibited TCA circulation was also observed through untargeted metabolomics. In addition, energy-related aspartate metabolism and lysine degradation were suppressed. In summary, these results indicated that IA and DI reduced bacterial pathogenicity while exerting antibacterial functions by inhibiting TCA circulation. This study enriches knowledge on the inhibition of bacteria by IA and DI in a carbon-mixed environment, suggesting an alternative method for treating bacterial infections by immune metabolites.

Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein.

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products - Rv1915 and Rv1916 - due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and 1 H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.

Sodium and lithium exert differential effects on the central carbon metabolism of Debaryomyces hansenii through the glyoxylate shunt regulation.

Debaryomyces hansenii is a halotolerant/halophilic yeast usually found in salty environments. The yeast accumulated sodium at high concentrations, which improved growth in salty media. In contrast, lithium was toxic even at low concentrations and its presence prevented cell proliferation. To analyse the responses to both cations, metabolite levels, enzymatic activities and gene expression were determined, showing that NaCl and LiCl trigger different cellular responses. At high concentrations of NaCl (0.5 or 1.5 M) cells accumulated higher amounts of the intermediate metabolites glyoxylate and malate and, at the same time, the levels of intracellular oxoglutarate decreased. Additionally, 0.5 M NaCl increased the activity of the enzymes isocitrate lyase and malate synthase involved in the synthesis of glyoxylate and malate respectively and decreased the activity of isocitrate dehydrogenase. Moreover, transcription of the genes coding for isocitrate lyase and malate synthase was activated by NaCl. Also, cells accumulated phosphate upon NaCl exposure. None of these effects was provoked when LiCl (0.1 or 0.3 M) was used instead of NaCl. Lithium induced accumulation of higher amounts of oxoglutarate and decreased the concentrations of glyoxylate and malate to non-detectable levels. Cells incubated with lithium also showed higher activity of the isocitrate dehydrogenase and neither increased isocitrate lyase and malate synthase activities nor the transcription of the corresponding genes. In summary, we show that sodium, but not lithium, up regulates the shunt of the glyoxylic acid in D. hansenii and we propose that this is an important metabolic adaptation to thrive in salty environments.

Arbuscular mycorrhiza differentially adjusts central carbon metabolism in two contrasting genotypes of Vigna radiata (L.) Wilczek in response to salt stress

The study aimed at investigating Arbuscular Mycorrhiza (AM) mediated metabolic changes in two genotypes of mungbean (Vigna radiata) differing in their salt tolerance in presence of salt stress (100 mM NaCl). Colonisation by Claroideoglomus etunicatum resulted in higher growth, photosynthetic efficiency, total protein content, and lower levels of stress markers, indicating alleviation of stress in mungbean plants. AM differentially upregulated the components of Tricarboxylic acid (TCA) cycle in salt tolerant (ST) and salt sensitive (SS) genotypes that could be correlated to AM-mediated moderation in nutrient uptake. Under salt stress, while maximum increase in the activity of α-ketoglutarate dehydrogenase (65%) was observed in mycorrhizal (M)-ST; the increase in isocitrate dehydrogenase (79%) and fumarase (133%) activities was maximum in M-SS plants over their non-mycorrhizal (NM) counterparts. Apart from TCA, AM also affected gamma-aminobutyric acid (GABA) and glyoxylate pathways. Activities of enzymes implicated in GABA shunt increased in both the genotypes under stress resulting in increase in GABA concentration (46%). Notably, glyoxylate pathway was induced by AM in SS only, wherein M-SS exhibited significantly higher isocitrate lyase (49%) and malate synthase (104%) activities, reflected in higher malic acid concentration (84%), than NM under stress. The results suggest that AM moderates the central carbon metabolism and strategizes towards boosting the formation of stress-alleviating metabolites such as GABA and malic acid, especially in SS, bypassing the steps catalysed by salt-sensitive enzymes in TCA cycle. The study, therefore, advances the understanding on mechanisms by which AM ameliorates salt stress.

Mechanisms of promotion in the heterotrophic growth of Chlorella vulgaris by the combination of sodium acetate and hydrolysate of broken rice

In order to reduce the culture cost and increase the growth rate of heterotrophic Chlorella vulgaris, the effects of hydrolysate of broken rice (HBR) combined with sodium acetate on its growth were evaluated. Results showed that the addition of 0.4 g/L of sodium acetate could stabilize the pH of the medium via the co-metabolism of acetate, ammonia and nitrate by Chlorella vulgaris. Meanwhile, isocitrate lyase activity increased threefold, which further promoted the glyoxylate cycle and the citric acid cycle, which finally provided more energy and metabolic precursors for cell growth. The biomass production (5.04 g/L), biomass productivity (1.65 g/L/day) and protein content (64.14 %) were 1.56, 1.81 and 1.77 times higher than the glucose group. This study demonstrated that HBR combined with sodium acetate could effectively promote the heterotrophic metabolism of microalgae, which provided scientific basis and guidance for industrial production of high-value products using Chlorella vulgaris as a fermentation platform.

Germination and the Biochemical Response of Pumpkin Seeds to Different Concentrations of Humic Acid under Cadmium Stress

The poisoning of heavy metals and their accumulation in food chains are major environmental and health risks. There have been several reports that determined that pumpkins tend to collect small amounts of nitrate or heavy metals. Therefore, the aim of the present study is to investigate the effect of organic matter (humic acid) on the germination and activity of antioxidant enzymes, glycosylate cycle enzymes, and utilization of lipid and protein reserves of pumpkin seeds under cadmium stress conditions. An experiment was conducted to quantify the germination response and biochemical change of pumpkin seeds to the use of humic acid under cadmium stress conditions. The treatments were cadmium at three levels (0 (control), 100, and 200 mg.L−1) and humic acid at five levels (0 (control), 100, 200, 300, and 400 mg.L−1). Linear and sigmoidal models were used to investigate the trend of trait changes. The results show that changes in the germination percentage and seed vigor were affected by applying humic acid and cadmium stress. The highest germination percentage for pumpkins was observed without stress and cadmium stress at a concentration of 200 mg.L−1. The results of quantification for the germination and seed vigor also showed that the model of germination changes by the use of humic acid was sigmoidal in non-stress and cadmium stress conditions of 100 mg.L−1, but it was linear for seed vigor in the stress conditions of 200 mg.L−1. The activity of superoxide dismutase, catalase, peroxidase, isocitrate lyase, and malate synthase was also affected by the simultaneous use of humic acid and cadmium stress, and the trend of their changes was linear.

D319 induced antifungal effects through ROS-mediated apoptosis and inhibited isocitrate lyase in Candida albicans

BackgroundCandida albicans (C. albicans) is an opportunistic pathogen that can cause superficial and life-threatening systemic infections in immunocompromised patients. However, the available clinically antifungals are limited. Therefore, the development of effective antifungal agents and therapies is urgently needed. Quinoline type of compounds were reported to possess potent anti-fungal effect. A series of quinoline derivatives were synthesized. Moreover their inhibitory activities and mechanisms on C. albicans were evaluated in this study. MethodsThe structure of D319 was identified by extensive spectroscopic analysis. The antifungal activity of D319 on C. albicans was evaluated using conventional methods, including the inhibition zone diameters with filter paper, Clinical Laboratory Standard Institute (CLSI) broth microdilution method in vitro, and in a murine model in vivo. Flow cytometry, fluorescence microscopy, western blot, knockout mutant and revertant strain techniques, and molecular modeling were applied to explore the mechanism of action of D319 in anti-Candida. ResultsD319 exhibited potent anti-Candida activity with Minimum Inhibitory Concentration value of 2.5 μg/mL in vitro. D319 significantly improved survival rate and reduced fungal burden compared to vehicle control in a murine model in vivo. The treatment of C. albicans with D319 resulted in fungal apoptosis through reactive oxygen species (ROS) accumulation in C. albicans. Furthermore, D319 inhibited the glyoxylate enzyme isocitrate lyase (ICL) of C. albicans, which was also confirmed by docking analysis. ConclusionsQuinoline compound D319 exhibited strong anti-Candida activities in vitro and in vivo models through inhibiting ICL activity and ROS accumulation in C. albicans. General significanceThis study showed that compound D319 as a novel isocitrate lyase inhibitor, would be a promising anti-Candida lead compound, which provided a potential application of this type of compounds in fighting clinical fungal infections. Furthermore, this study also supported ICL as a potential target for anti-Candida drug discovery.

Hydro-Electro Hybrid Priming Promotes Carrot (Daucus carota L.) Seed Germination by Activating Lipid Utilization and Respiratory Metabolism.

Carrot (Daucus carota L.) is widely cultivated as one of the most important root crops, and developing an effective presowing treatment method can promote the development of modern mechanized precision sowing. In the present study, a novel seed priming technology, named hydro-electro hybrid priming (HEHP), was used to promote the germination of carrot seeds. Seed germination experiments showed that HEHP was able to increase the germination index (GI) and vigor index (VI) by 3.1-fold and 6.8-fold, respectively, and the effect was significantly superior to that of hydro-priming (HYD) and electrostatic field treatment (EF). The consumption and utilization rate of seed storage reserves were also greatly improved. Meanwhile, both glyoxysomes and mitochondria were found to appear ahead of time in the endosperm cells of HEHP through observations of the subcellular structure of the endosperm. Activities of isocitrate lyase (ICL), NAD-dependent malate dehydrogenase (MDH), pyruvate kinase (PK), and alcohol dehydrogenase (ADH) were significantly increased by HEHP. From transcriptome results, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the glyoxylate cycle, glycolysis, gluconeogenesis, and the citrate cycle were significantly enriched and real-time quantitative PCR (qRT-PCR) analysis confirmed the expression pattern of 15 critical differentially expressed genes (DEGs) in these pathways. All DEGs encoding MDH, phosphoenolpyruvate carboxykinase (PEPCK), and PK were upregulated in HEHP; thus, it is reasonable to infer that the transformation of malate, oxalacetate, phosphoenolpyruvate, and pyruvate in the cytoplasm may be pivotal for the energy supply during early germination. The results suggest that the optimal effect of HEHP is achieved by initiating stored lipid utilization and respiratory metabolism pathways related to germination.

60Coγ-ray irradiation inhibits germination of fresh walnuts by modulating respiratory metabolism and reducing energy status during storage

Postharvest germination negatively affects the development of fresh walnut industry. Our previous research showed that 60Coγ-ray irradiation with 0.3 kGy effectively inhibited the germination of fresh walnuts during storage, but the underlying mechanism in respiratory metabolism remains still unclear. The untreated and 0.3 kGy-irradiated fresh walnuts were stored at 25 °C for 24 d, and then the indicators related to respiratory metabolism in kernels and embryos were determined. The results found that the 0.3 kGy irradiation decreased the respiration intensity after 15d and completely inhibited the germination of fresh walnuts with the declined adenosine triphosphate (ATP) content and energy charge (EC) levels in kernels except from 12 to 15 d and polyphenol oxidase (PPO), cytochrome C oxidase (CCO) and ascorbic acid oxidase (AAO) activities in both tissues except the stimulated PPO activity in embryos during storage. Moreover, the irradiation promoted the glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) activities but reduced the isocitrate lyase (ICL) activity in both tissues, and decreased the nicotinamide adenine dinucleotide reduced form (NADH) content but increased the nicotinamide adenine dinucleotide phosphate reduced form (NADPH) content in kernels of fresh walnuts during storage. The irradiation induced the JRG6PDH and JR6PGDH expression levels in both tissues, as well as the JRICL and JRMS expression levels in kernels before 12 d but suppressed those after 12 d of storage. In conclusion, the germination inhibition of fresh walnuts by gamma irradiation is attributed to suppressed terminal oxidases activities, decreased ratio of Embden–Meyerhof–Parnas pathway-tricarboxylic acid cycle (EMP-TCA) and glyoxylate cycle (GAC), increased level of pentose phosphate pathway (PPP) and reduced energy status.

Substrate usage determines carbon flux via the citrate cycle in Helicobacter pylori

Helicobacter pylori displays a worldwide infection rate of about 50%. The Gram-negative bacterium is the main reason for gastric cancer and other severe diseases. Despite considerable knowledge about the metabolic inventory of H. pylori, carbon fluxes through the citrate cycle (TCA cycle) remained enigmatic. In this study, different 13 C-labeled substrates were supplied as carbon sources to H. pylori during microaerophilic growth in a complex medium. After growth, 13 C-excess and 13 C-distribution were determined in multiple metabolites using GC-MS analysis. [U-13 C6 ]Glucose was efficiently converted into glyceraldehyde but only less into TCA cycle-related metabolites. In contrast, [U-13 C5 ]glutamate, [U-13 C4 ]succinate, and [U-13 C4 ]aspartate were incorporated at high levels into intermediates of the TCA cycle. The comparative analysis of the 13 C-distributions indicated an adaptive TCA cycle fully operating in the closed oxidative direction with rapid equilibrium fluxes between oxaloacetate-succinate and α-ketoglutarate-citrate. 13 C-Profiles of the four-carbon intermediates in the TCA cycle, especially of malate, together with the observation of an isocitrate lyase activity by in vitro assays, suggested carbon fluxes via a glyoxylate bypass. In conjunction with the lack of enzymes for anaplerotic CO2 fixation, the glyoxylate bypass could be relevant to fill up the TCA cycle with carbon atoms derived from acetyl-CoA.

Progesterone Promotes Mitochondrial Respiration at the Biochemical and Molecular Level in Germinating Maize Seeds

This research aimed to investigate the effects of progesterone, a mammalian steroid sex hormone, on the mitochondrial respiration in germinating maize seeds. For this purpose, maize seeds were divided into four different groups (control, 10−6, 10−8, and 10−10 mol·L−1 progesterone) and were grown in a germination cabinet in the dark at 24.5 ± 0.5 °C for 4 d. The changes in gene expression levels of citrate synthase (CS), cytochrome oxidase (COX19), pyruvate dehydrogenase (Pdh1), and ATP synthase (ATP6), which is involved in mitochondrial respiration, were studied in root and cotyledon tissues. Significant increases were recorded in the gene expression levels of all studied enzymes. In addition, progesterone applications stimulated activities of malate synthase (MS), isocitrate lyase (ICL), and alpha-amylase, which are important enzymes of the germination step. The changes in gene expression levels of mas1 and icl1 were found parallel to the rise in these enzymes’ activities. It was determined similar increases in root and coleoptile lengths and total soluble protein and total carbohydrate contents. The most remarkable changes were detected in 10−8 mol·L−1 progesterone-treated seedlings. These results clearly indicate that progesterone stimulates mitochondrial respiration by inducing biochemical and molecular parameters and thus accelerates seed germination thanks to the activation of other pathways related to mitochondrial respiration.

RegX3 Controls Glyoxylate Shunt and Mycobacteria Survival by Directly Regulating the Transcription of Isocitrate Lyase Gene in Mycobacterium smegmatis.

The glyoxylate shunt is a pathway associated with the assimilation of fatty acids and is implicated in the resistance of M. tuberculosis (Mtb). Isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, mediates Mtb infections and its survival in mice via fatty acids, metabolism, and physiological functions. Here, we found that in Mycobacterium smegmatis (M. smegmatis) the two-component system SenX3-RegX3 regulated the glyoxylate shunt in response to phosphate starvation by controlling the transcription of icl. In response to phosphate availability, the phosphate regulator RegX3 directly bound to the upstream regulatory region of icl and repressed its transcription. The inactivation of regX3 increased icl transcription and ICL activity, causing a growth defect in M. smegmatis with fatty acids as the sole source of carbon and energy. The growth defect was partly due to the toxicity of the excess glyoxylate produced by ICL. A decrease in glyoxylic acid levels, overexpression of regX3, or the chemical inhibition (IA or 3-NP) of ICL restored the growth of the Regx3-deficient M. smegmatis. Thus, we established a genetic network between the phosphate stress response and glyoxylate shunt based on the amount of intracellular ICL during mycobacterial survival on short-chain fatty acids, which contributed to its antimicrobial arsenal.

Expression of the ace operon in Escherichia coli is triggered in response to growth rate-dependent flux-signal of ATP.

The signal that triggers the expression of the ace operon and, in turn, the transition of central metabolism's architecture from acetogenic to gluconeogenic in Escherichia coli remains elusive despite extensive research both in vivo and in vitro. Here, with the aid of flux analysis together with measurements of the enzymic activity of isocitrate lyase (ICL) and its aceA-messenger ribonucleuc acid (mRNA) transcripts, we provide credible evidence suggesting that the expression of the ace operon in E. coli is triggered in response to growth rate-dependent threshold flux-signal of adenosine triphosphate (ATP). Flux analysis revealed that the shortfall in ATP supply observed as the growth rate ($\mu $) diminishes from µmax to≤0.43h-1 ($ \pm 0.02;n4)\ $is partially redressed by up-regulating flux through succinyl CoA synthetase. Unlike glycerol and glucose, pyruvate cannot feed directly into the two glycolytic ATP-generating reactions catalyzed by phosphoglycerokinase and pyruvate kinase. On the other hand, glycerol, which upon its conversion to D-glyceraldehyde, feeds into the phosphorylation and dephosphorylation parts of glycolysis including the substrate-level phosphorylation-ATP generating reactions, thus preventing ATP flux from dropping to the critical threshold signal required to trigger the acetate-diauxic switch until glycerol is fully consumed. The mRNA transcriptional patterns of key gluconeogenic enzymes, namely, ackA, acetate kinase; pta, phosphotransacetylase; acs, acetyl CoA synthetase and aceA, ICL, suggest that the pyruvate phenotype is better equipped than the glycerol phenotype for the switch from acetogenic to gluconeogenic metabolism.

A Biostimulant Seed Treatment Improved Heat Stress Tolerance During Cucumber Seed Germination by Acting on the Antioxidant System and Glyoxylate Cycle.

Seed enhancement technologies have the potential to improve germination and seedling growth under environmental stress. The effects of KIEM®, an innovative biostimulant based on lignin derivatives and containing plant-derived amino acids and molybdenum, were investigated on cucumber (Cucumis sativus L.) seed germination. To determine the metabolic targets of this product, biometric, transcriptional and biochemical analyses were carried out on both non-treated and KIEM®-treated seeds incubated for 24 and 48 h under standard (28°C) and heat stress (35°C) conditions. The application of the biostimulant as a seed treatment increased the percent germination (+6.54%) and fresh biomass (+13%) at 48 h, and decreased the content of H2O2 in treated seeds at 28°C (−70%) and at 35°C (−80%). These changes in biometric and biochemical properties were accompanied by changes in expression levels of the genes coding for ROS-producing (RBOH) and scavenging (SOD, CAT, GST) enzymes and their specific activity. In general, the treatment with KIEM® in heat-stress condition appeared to stimulate a higher accumulation of three scavenger gene transcripts: CuZnSOD (+1.78), MnSOD (+1.75), and CAT (+3.39), while the FeSOD isoform was dramatically downregulated (0.24). Moreover, the amount of non-protein thiols, important antioxidant molecules, was increased by the biostimulant after 48 h (+20%). Taken together these results suggest that KIEM® acts through mitigation of the effects of the oxidative stress. Moreover, after 48 h, the pre-sowing treatment with KIEM® increased the transcription levels (+1.5) and the activity of isocitrate lyase (+37%), a key enzyme of the glyoxylate cycle, suggesting a potential effect of this product in speeding up the germination process. Finally, the chemical characterization of KIEM® identified five essential and three non-essential amino acids, and others bioactive compounds, including five organic and inorganic acids that might be potentially involved in its activity. Based on these data, insights on the potential mechanism of action of the biostimulant, suggested that there are broader applications as a product able to increase seed tolerance to different abiotic stress typical of adverse environmental conditions.