EuHDZ25 positively affects rubber biosynthesis by targeting EuFPS1 in Eucommia leaves.

Eucommia ulmoides is a temperate gum source plant that produces trans-polyisoprene (TPI), also known as Eucommia rubber. The structural configuration and function of TPI offer a new material with important potential for industrial development. In this study, we detected the TPI content in the leaves of diploid and triploid E. ulmoides plants. The average TPI content in the leaves of triploid E. ulmoides was significantly higher than that of diploid. Transcriptome data and weighted gene co-expression network analyses identified a significant positive correlation between the EuFPS1 gene and TPI content. Overexpression of EuFPS1 increased the density of rubber particles and TPI content, indicating its crucial role in TPI biosynthesis. In addition, the expression of EuHDZ25 in E. ulmoides was significantly positively correlated with EuFPS1 expression. Yeast one-hybrid and dual-luciferase assays demonstrated that EuHDZ25 mainly promotes TPI biosynthesis through positive regulation of EuFPS1 expression. The significantly up-regulated expression of EuHDZ25 and its consequent upregulation of EuFPS1 during the biosynthesis of TPI may partially explain the increased TPI content of triploids. This study provides an important theoretical foundation for further exploring the molecular mechanism of secondary metabolites content variation in polyploids and can help to promote the development and utilization of rubber resources.

Computational investigation of cis-1,4-polyisoprene binding to the latex-clearing protein LcpK30.

Latex clearing proteins (Lcps) catalyze the oxidative cleavage of the C = C bonds in cis-1,4-polyisoprene (natural rubber), producing oligomeric compounds that can be repurposed to other materials. The active catalytic site of Lcps is buried inside the protein structure, thus raising the question of how the large hydrophobic rubber chains can access the catalytic center. To improve our understanding of hydrophobic polymeric substrate binding to Lcps and subsequent catalysis, we investigated the interaction of a substrate model containing ten carbon-carbon double bonds with the structurally characterized LcpK30, using multiple computational tools. Prediction of the putative tunnels and cavities in the LcpK30 structure, using CAVER-Pymol plugin 3.0.3, fpocket and Molecular Dynamic (MD) simulations provided valuable insights on how substrate enters from the surface to the buried active site. Two dominant tunnels were discovered that provided feasible routes for substrate binding, and the presence of two hydrophobic pockets was predicted near the heme cofactor. The larger of these pockets is likely to accommodate the substrate and to determine the size distribution of the oligomers. Protein-ligand docking was carried out using GOLD software to predict the conformations and interactions of the substrate within the protein active site. Deeper insight into the protein-substrate interactions, including close-contacts, binding energies and potential cleavage sites in the cis-1,4-polyisoprene, were obtained from MD simulations. Our findings provide further justification that the protein-substrate complexation in LcpK30 is mainly driven by the hydrophobic interactions accompanied by mutual conformational changes of both molecules. Two potential binding modes were identified, with the substrate in either extended or folded conformations. Whilst binding in the extended conformation was most favorable, the folded conformation suggested a preference for cleavage of a central double bond, leading to a preference for oligomers with 5 to 6 C = C bonds. The results provide insight into further enzyme engineering studies to improve catalytic activity and diversify the substrate and product scope of Lcps.

Optimal seasonal schedule for the production of isoprene, a highly volatile biogenic VOC.

The leaves of many trees emit volatile organic compounds (abbreviated as BVOCs), which protect them from various damages, such as herbivory, pathogens, and heat stress. For example, isoprene is highly volatile and is known to enhance the resistance to heat stress. In this study, we analyze the optimal seasonal schedule for producing isoprene in leaves to mitigate damage. We assume that photosynthetic rate, heat stress, and the stress-suppressing effect of isoprene may vary throughout the season. We seek the seasonal schedule of isoprene production that maximizes the total net photosynthesis using Pontryagin's maximum principle. The isoprene production rate is determined by the changing balance between the cost and benefit of enhanced leaf protection over time. If heat stress peaks in midsummer, isoprene production can reach its highest levels during the summer. However, if a large portion of leaves is lost due to heat stress in a short period, the optimal schedule involves peaking isoprene production after the peak of heat stress. Both high photosynthetic rate and high isoprene volatility in midsummer make the peak of isoprene production in spring. These results can be clearly understood by distinguishing immediate impacts and the impacts of future expectations.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Histochemical studies on the distribution and accumulation of trans-polyisoprene in the rubber-producing plant Eucommia ulmoides.

Eucommia ulmoides rubber (EUR) is a high-quality natural rubber resource, which can be extracted from different organs of the Eucommia ulmoides tree. In this study, EUR was isolated from the leaves, barks, and pericarps, and the structural characteristics and physicochemical properties of EUR were systematically determined. The accumulation and distribution of EUR in different tissues were assessed through in situ observations combined with cellular and subcellular scales. The preliminary analyses indicated that the variations in the physicochemical properties of EUR across different tissues were associated with its accumulation microstructure. Further analyses by SEM and TEM showed that the initial cell differentiation and fusion resulted in the formation of tubular structures without any nucleus. A limited number of rubber particles were generated within the cytoplasm, concurrent with aggregation and fusion. Eventually, rubber particles filled the entire cytoplasm, and organelles disappeared to form highly aggregated filamentous structures. In addition, the number and area of EUR-containing cells were closely related to the organization sizes of barks and leaves. This study provided valuable insights into Eucommia ulmoides histology and the rubber industry.

Chemical Composition, Anti-bacterial Activity and Molecular Docking Studies of Essential Oil Isolated from Sa Sam Nam (Launaea sarmentosa).

Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.

Isoprenoid emissions from Schima superba and Cunninghamia lanceolata: Their responses to elevated temperature by two warming facilities.

Isoprenoids (including isoprene (ISO) and monoterpenes (MTs)) are the majority of biogenic volatile organic compounds (BVOCs) which are important carbon-containing secondary metabolites biosynthesized by organisms, especially plant in terrestrial ecosystem. Results of the warming effects on isoprenoid emissions vary within species and warming facilities, and thus conclusions remain controversial. In this study, two typical subtropical tree species seedlings of Schima superba and Cunninghamia lanceolata were cultivated under three conditions, namely no warming (CK) and two warming facilities (with infrared radiators (IR) and heating wires (HW)) in open top chamber (OTC), and the isoprenoid emissions were measured with preconcentor-GC-MS system after warming for one, two and four months. The results showed that the isoprenoid emissions from S. superba and C. lanceolata exhibited uniformity in response to two warming facilities. IR and HW both stimulated isoprenoid emissions in two plants after one month of treatment, with increased ratios of 16.3 % and 72.5 % for S. superba, and 2.47 and 5.96 times for C. lanceolata. However, the emissions were suppressed after four months, with more pronounced effect for HW. The variation in isoprenoid emissions was primarily associated with the levels of Pn, Tr, monoterpene synthase (MTPS) activity. C. lanceolata predominantly released MTs (mainly α-pinene, α-terpene, γ-terpene, and limonene), with 39.7 % to 99.6 % of the total isoprenoid but ISO was only a very minor constituent. For S. superba, MTs constituted 24.7 % to 96.1 % of total isoprenoid. It is noteworthy that HW generated a greater disturbance to physiology activity in plants. Our study provided more comprehensive and more convincing support for integrating temperature-elevation experiments of different ecosystems and assessing response and adaptation of forest carbon cycle to global warming.

Unravelling the origin of isoprene in the human body-a forty year Odyssey.

In the breath research community's search for volatile organic compounds that can act as non-invasive biomarkers for various diseases, hundreds of endogenous volatiles have been discovered. Whilst these systemic chemicals result from normal and abnormal metabolic activities or pathological disorders, to date very few are of any use for the development of clinical breath tests that could be used for disease diagnosis or to monitor therapeutic treatments. The reasons for this lack of application are manifold and complex, and these complications either limit or ultimately inhibit the analytical application of endogenous volatiles for use in the medical sciences. One such complication is a lack of knowledge on the biological origins of the endogenous volatiles. A major exception to this is isoprene. Since 1984, i.e. for 40 years, it has been generally accepted that the pathway to the production of human isoprene, and hence the origin of isoprene in exhaled breath, is through cholesterol biosynthesis via the mevalonate (MVA) pathway within the liver. However, various studies between 2001 and 2012 provide compelling evidence that human isoprene is produced in skeletal muscle tissue. A recent multi-omic investigation of genes and metabolites has revealed that this proposal is correct by showing that human isoprene predominantly results from muscular lipolytic cholesterol metabolism. Despite the overwhelming proof for a muscular pathway to isoprene production in the human body, breath research papers still reference the hepatic MVA pathway. The major aim of this perspective is to review the evidence that leads to a correct interpretation for the origins of human isoprene, so that the major pathway to human isoprene production is understood and appropriately disseminated. This is important, because an accurate attribution to the endogenous origins of isoprene is needed if exhaled isoprene levels are to be correctly interpreted and for assessing isoprene as a clinical biomarker.

Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar.

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

Quantifying exhaled acetone and isoprene through solid phase microextraction and gas chromatography-mass spectrometry.

BACKGROUND: Acetone, isoprene, and other volatile organic compounds (VOCs) in exhaled breath have been shown to be biomarkers for many medical conditions. Researchers use different techniques for VOC detection, including solid phase microextraction (SPME), to preconcentrate volatile analytes prior to instrumental analysis by gas chromatography-mass spectrometry (GC-MS). These techniques include a previously developed method to detect VOCs in breath directly using SPME, but it is uncommon for studies to quantify exhaled volatiles because it can be time consuming due to the need of many external/internal standards, and there is no standardized or widely accepted method. The objective of this study was to develop an accessible method to quantify acetone and isoprene in breath by SPME GC-MS. RESULTS: A system was developed to mimic human exhalation and expose VOCs to a SPME fiber in the gas phase at known concentrations. VOCs were bubbled/diluted with dry air at a fixed flow rate, duration, and volume that was comparable to a previously developed breath sampling method. Identification of acetone and isoprene through GC-MS was verified using standards and observing overlaps in chromatographic retention/mass spectral fragmentation. Calibration curves were developed for these two analytes, which showed a high degree of linear correlation. Acetone and isoprene displayed limits of detection/quantification equal to 12 ppb/37 ppb and 73 ppb/222 ppb respectively. Quantification results in healthy breath samples (n = 15) showed acetone concentrations spanned between 71 ppb and 294 ppb, and isoprene varied between 170 ppb and 990 ppb. Both concentration ranges for acetone and isoprene in this study overlap with those reported in existing literature. SIGNIFICANCE: Results indicate the development of a system to quantify acetone and isoprene in breath that can be adapted to diverse sampling methods and instrumental analyses beyond SPME GC-MS.

Molecular cloning and characterization of farnesyl diphosphate synthase from Rosa rugosa Thunb associated with salinity stress.

Rosa rugosa, a renowned ornamental plant, is cultivated for its essential oil containing valuable monoterpenes, sesquiterpenes, and other compounds widely used in the floriculture industry. Farnesyl diphosphate synthase (FPPS) is a key enzyme involved in the biosynthesis of sesquiterpenes and triterpenes for abiotic or biotic stress. In this study, we successfully cloned and characterized a full-length FPPS- encoding cDNA identified as RrFPPS1 using RT-PCR from R. rugosa. Phylogenetic analysis showed that RrFPPS1 belonged to the angiosperm-FPPS clade. Transcriptomic and RT-qPCR analyses revealed that the RrFPPS1 gene had tissue-specific expression patterns. Subcellular localization analysis using Nicotiana benthamiana leaves showed that RrFPPS1 was a cytoplasmic protein. In vitro enzymatic assays combined with GC-MS analysis showed that RrFPPS1 produced farnesyl diphosphate (FPP) using isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to provide a precursor for sesquiterpene and triterpene biosynthesis in the plant. Additionally, our research found that RrFPPS1 was upregulated under salt treatment. These substantial findings contribute to an improved understanding of terpene biosynthesis in R. rugosa and open new opportunities for advancements in horticultural practices and fragrance industries by overexpression of the RrFPPS1 gene in vivo increased FPP production and subsequently led to elevated sesquiterpene yields in the future. The knowledge gained from this study can potentially lead to the development of enhanced varieties of R. rugosa with improved aroma, medicinal properties, and resilience to environmental stressors.

Mixed Potential Type Isoprene Sensor for the Application in Real-Time Monitoring of Biomarker Gases.

Monitoring of isoprene in exhaled breath is expected to provide a noninvasive and painless method for dynamic monitoring of physiological and metabolic states during exercise. However, for real-time and portable detection of isoprene, gas sensors have become the best choice for gas detection technology, which are crucial to achieving the goal of anytime, anywhere, human-centered healthcare in the future. Here, we first report a mixed potential type isoprene sensor based on a Gd2Zr2O7 solid electrolyte and a CdSb2O6 sensing electrode, which enables sensitive detection for isoprene with sensitivities of -21.2 mV/ppm and -65.8 mV/decade in the range of 0.05-1 and 1-100 ppm. The sensing behavior of the sensor follows the mixed potential sensing mechanism and was further verified by the electrochemical polarization curves. The significant differentiation between the sensor response to exhaled breath of healthy individuals and simulated breath containing different concentrations of isoprene demonstrates the potential of the sensor for the detection of isoprene in exhaled breath. Simultaneously, monitoring of isoprene during exercise signifies the feasibility of the sensor in dynamic monitoring of physiological indicators, which is not only of great significance for optimizing training and guiding therapeutic exercise intervention in sporting scenarios but also expected to help further reveal the interaction between exercise, muscle, and organ metabolism in medicine.

Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity.

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species (Suillus hirtellus EM16, Suillus decipiens EM49, and Suillus cothurnatus VC1858), core members of the pine microbiome. After 28 days of growth on solid media, liquid chromatography-tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 41 putative prenol lipids (includes 37 putative terpenes) were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication.IMPORTANCEUsing a combination of genomics and metabolomics, this study's findings offer new insights into the chemical diversity of Suillus fungi, which serve a critical role in forest ecosystems.

Metabolomic insights into the browning inhibition of fresh-cut apple by hydrogen sulfide.

Hydrogen sulfide (H2S) is known to effectively inhibit the browning of fresh-cut apples, but the mechanism at a metabolic level remains unclear. Herein, non-targeted metabolomics was used to analyze metabolic changes in surface and internal tissues of fresh-cut apple after H2S treatment. The results showed that prenol lipids were the most up-accumulated differential metabolites in both surface and inner tissue of fresh-cut apple during browning process, which significantly down-accumulated by H2S treatment. H2S treatment reduced the consumption of amino acid in surface tissue. Regarding inner tissue, H2S activated defense response through accumulation of lysophospholipid signaling and induced the biosynthesis of phenolic compounds. We therefore propose that H2S inhibited the surface browning of fresh-cut apple by reducing the accumulation of prenol lipids, directly delaying amino acid consumption in surface tissue and indirectly regulating defense response in inner tissue, which provides fundamental insights into browning inhibition mechanisms by H2S.

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

Functional characterization of a geranylgeranyl diphosphate synthase in the leaf beetle Monolepta hieroglyphica.

Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.

Therapeutic effect of Sanhua decoction on rats with middle cerebral artery occlusion and the associated changes in gut microbiota and short-chain fatty acids.

Sanhua decoction (SHD), a traditional prescription, has long been used in treating ischemic stroke (IS). However, the therapeutic effect of SHD and the associated changes in gut microbiota and short-chain fatty acids (SCFAs) are uncertain. In this study, a rat model of IS was established by the middle cerebral artery occlusion (MCAO). By evaluating the cerebral infarct area and brain tissue pathology, it was found that SHD ameliorated IS-related symptoms in MCAO rats. Using 16S rRNA gene sequencing, we found that SHD reduced abnormally elevated Lactobacillus and opportunistic pathogens such as Desulfovibrio, but increased some beneficial bacteria that produce SCFAs, including Clostridia, Lachnospiraceae, Ruminococcaceae, and Coprococcus. KEGG analysis revealed that SHD regulates several pathways, including D-arginine and D-ornithine metabolism, polyketide sugar unit biosynthesis, and cyanoamino acid metabolism, which are significantly altered in MCAO rats. By gas chromatography-mass spectrometry detection of SCFAs, we found that fecal acetic acid, valeric acid, and caproic acid were significantly increased in MCAO rats, whereas propionic acid and isobutyric acid were decreased. SHD reversed the changes in acetic acid and propionic acid in the model rats and significantly increased fecal butyric acid. In addition, MCAO rats had significantly higher serum levels of acetic acid, butyric acid, isovaleric acid, and valeric acid, and lower levels of caproic acid. Altered serum levels of butyric acid, isovaleric acid, valeric acid, and caproic acid were restored, and the level of isobutyric acid was reduced after SHD administration. Spearman analysis revealed that cerebral infarct area had a strong correlation with Bifidobacterium, Desulfovibrio, Lachnospiraceae, Lactobacillus, acetic acid, valeric acid, and caproic acid. Overall, this study demonstrates for the first time that the effect of SHD on IS may be related to gut microbiota and SCFAs, providing a potential scientific explanation for the ameliorative effect of SHD on IS.

Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites.

A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.

Polar Molecules Regulating the Regio- and Stereoselectivity of Polymerization of Conjugated Dienes Catalyzed by CGC-Type Rare-Earth Metal Catalysts.

Herein, a concise, effective, and scalable strategy is reported that the introduction of polar molecules (PMs) (e.g., anisole (PhOMe), phenetole (PhOEt), 2-methoxynaphthalene (NaphOMe), thioanisole (PhSMe), and N,N-dimethylaniline (PhNMe2)) as continuously coordinated neutral ligand of cationic active species in situ generated from the constrain-geometry-configuration-type rare-earth metal complexes A-F/AliBu3/[Ph3C][B(C6F5)4] ternary systems can easily switch the regio- and stereoselectivity of the polymerization of conjugated dienes (CDs, including 2-subsituted CDs such as isoprene (IP) and myrcene (MY), 1,2-disubstituted CD ocimene (OC), and 1-substituted polar CD 1-(para-methoxyphenyl)-1,3-butadiene (p-MOPB)) from poor selectivities to high selectivities (for IP and MY: 3,4-selectivity up to 99%; for OC: trans-1,2-selectivity up to 93% (mm up to 90%); for p-MOPB: 3,4-syndioselectivity (3,4- up to 99%, rrrr up to 96%)). DFT calculations explain the continuous coordination roles of PMs on the regulation of the regio- and stereoselectivity of the polymerization of CDs. In comparison with the traditional strategies, this strategy by adding some common PMs is easier and more convenient, decreasing the synthetic cost and complex operation of new metal catalyst and cocatalyst. Such regio- and stereoselective regulation method by using PMs is not reported for the coordination polymerization of olefins catalyzed by rare-earth metal and early transition metal complexes.