Chorismate Synthase Research Articles

Microplastics mitigate the effects of glyphosate on the shikimic acid pathway enzymes in cyanobacteria

Microplastics, an environmental contaminant, can directly harm aquatic organisms and alter the toxicity and availability of other pollutants. The herbicide glyphosate (N-(phosphonomethyl) glycine) exerts its toxicity by inhibiting 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), a key enzyme belonging to shikimic acid pathway that leads to the biosynthesis of aromatic amino acids; tolerance, instead, is dependent on a glyphosate -resistant EPSPS. We tested the effects of 0,2mM glyphosate alone and in combination with 170 mg/mL polyethylene microplastics (MPs) on the expression of EPSPS and chorismate synthase (ChS) on a culture of the cyanobacterium Limnospira maxima. Glyphosate caused the highest mortality after one week, with the resistant population surviving thereafter.However, when the culture was exposed to the combined treatment the mortality decreased. Proteomic profiles of treated cultures demonstrated that both enzymes were accumulated during the 21 days glyphosate treatment, suggesting that the surviving population of L. maxima might express glyphosate-resistant target enzymes. Treatment with combined solution caused a decrease of EPSPS and ChS levels compared to those of glyphosate alone. Our results strongly suggest that microplastics mitigate the toxicity of glyphosate towards target enzymes of the shikimic acid pathway, thereby increasing the resistance capacity of the L. maxima strain.

The mechanism of low temperature enhances dough aromas during fermentation by Pichia kudriavzevii: Sugar and amino acid metabolism, acid utilization and alcohol production

In order to investigate the impact of low temperature on enhancing aromas in fermented dough by Pichia kudriavzevii CGMCC 17607 (EP), the study initially utilized gas chromatography to detect volatile organic compounds (VOCs), followed by analyzing basal metabolites derived from these VOCs. Metabolomics was employed to monitor metabolic changes during fermentation, while proteomics was used to examine protein expression related to these changes. Results indicated notable increases in VOCs such as 3-methyl-1-butanol and 2-phenylethanol. Essential metabolites exhibited decreases in sugars (e.g., sucrose, maltose), organic acids (e.g., lactic acid, acetic acid), and amino acids (e.g., leucine, histidine). Particularly, low temperature fermentation led to raised levels of NAD+, NADH, acyltransferases, and ATP, excluding pyruvic acid and glucose-6-phosphate dehydrogenase. These outcomes suggested that low temperature fermentation boosted acid utilization and alcohol production. Metabolomics and proteomics highlighted decreased glycolysis influencing energy metabolism. Moreover, metabolomics revealed elevated levels of flavor substances like octanoic acid and its derivatives with aromatic properties, as well as geranic acid with terpenoid aroma. Furthermore, key intermediates like chorismite and phenylpyruvic acid, along with the pivotal enzyme chorismate synthase, illustrated the biosynthesis pathway of shikimic acid instead of phenylalanine. In summary, at low temperature, EP showed weakened glycolysis, with sugars and amino acids metabolizing into desirable flavor substances like alcohols, which promoted acidity reduction and aroma enhancement of dough.

258 Investigating the Ruminal Metagenome of Neonatal Holstein Dairy Calves fed an Essential oil-Based Feed Additive

Abstract Antibiotics remain the most effective tool for treating and preventing animal diseases in livestock production. However, the widespread use of antibiotics in food animals, as well as concerns about antibiotic resistance among bacterial pathogens, have raised awareness of the need to limit their use. As neonatal dairy calves are very susceptible to gastrointestinal infections that can result in high mortality and morbidity, the need to find alternatives to antibiotics has become a priority. Although essential oils (EO) have emerged as promising anti-microbial alternatives, their potential effects on the rumen microbiome are still poorly understood due to the complexity of their chemical components and limited understanding of their mechanisms of action. To gain further insight into the effects of EO on the bacterial communities of pre-weaned calves, we investigated the rumen metagenome of neonatal Holstein dairy calves that had been supplemented with 3.75g/feeding of a blend of EO (carvacrol, caryophyllene, p-cymene, cineole, terpinene, and thymol) as part of a previous study. Genomic DNA extracted from rumen contents collected from one of the EO-supplemented animals was used for high throughput sequencing using an Illumina Miseq (2x250) platform. Raw sequence reads were assembled into genomic contigs using custom-written Perl scripts. Coding sequences (CDS) were identified and annotated using RAST, with assignment of open reading frames (ORFs) to metabolic pathways using KEGG pathways. After assembly, a total of 3,052 contigs between 279 and 259,890 nt in length were obtained. From this set, 571 contigs affiliated to Prevotella ruminicola, representing 18.7% of total contigs built, were further analyzed. Data mining for specific functions within categories such as ‘protein metabolism’, ‘carbohydrates metabolism’, and ‘amino acids and derivatives’ was performed. This analysis resulted in the identification of all the enzymes involved in glycolysis. Two outcomes were predicted for pyruvate: conversion to lactate through lactate dehydrogenases (EC 1.1.1.27, EC 1.1.1.28), and the production of formate and acetyl-CoA through the activity of pyruvate formate-lyase (EC 2.3.1.54). Acetate (acetate kinase, EC 2.7.2.1) and propionate (Propionate kinase, EC 2.7.2.15) were other predicted end products. In addition to glucose fermentation pathways, enzymes potentially involved in the utilization of plant-based secondary metabolites were also identified, such as enzymes involved in Quinate-Chorismate metabolism: 3-dehydroquinate dehydratase (EC 4.2.1.10), Shikimate 5-dehydrogenase I alpha (EC 1.1.1.25), Shikimate kinase (EC 2.7.1.71), 5-Enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), and Chorismate synthase (EC 4.2.3.5). Identification of such metabolic pathways suggests that perhaps the supplemented EO additive could be used as a precursor for the synthesis of aromatic amino acids. Together, these findings indicate that EO supplementation may benefit animal health and performance by promoting the growth of bacterial species that can metabolize compounds in EO additives.

Evidence of Bi-Directional Volatile-Mediated Communication between Drought-Stressed and Well-Watered Grapevines (Vitis vinifera L.)

The volatile-mediated interplay between stressed and non-stressed plants has been described in many studies involving both biotic and abiotic stresses as a one-way channel. However, very little is known about the molecular basis and mechanisms by which volatile organic compounds (VOCs) mediate plant communication between drought-stressed ‘emitter’ plants and non-stressed ‘receiver’ neighbours for the defence against impending stress challenges. Aiming to address this in grapevine, this study investigated the effect of two-way VOC exchange between stressed and non-stressed Vitis vinifera L. cv. Shiraz during drought and recovery using four treatments: isolated well-watered (WW) vines, isolated drought-stressed (DS) vines, and co-located DS ‘emitter’ and WW ‘receiver’ vines in a growth room. The results obtained from solid-phase microextraction (SPME) gas chromatography mass spectrometry (GC-MS) analysis showed a synchronised decline in α-pinene concentration in the co-located treatment vines and higher isoprene levels in the DS emitters compared to the isolated DS vines. Targeted gene expression analysis further identified the over-expression of a key gene, allene oxide synthase (AOS), in the jasmonic acid (JA) biosynthesis pathway during peak drought in the DS emitter. Transcript expression of chorismate synthase (CHORS) and α-pinene synthase (VvPNaPin1) showed similar trends in the DS emitter. The results suggest that isoprene and α-pinene may be interplant signalling molecules used by grapevine during drought. To the best of our knowledge, this is the first report of a bi-directional interaction in grapevine between the emitters and receivers under drought stress mediated by the JA and terpenoid biosynthesis pathways.

Disruption of CHORISMATE SYNTHASE1 leads to yellow-green variegation in soybean leaves.

Yellow-green variegation leaf phenotype adds more value to ornamental plants, but it is regarded as an undesirable trait in crop plants, affecting their yields. Until recently, the underlying mechanism regulating the yellow-green variegation phenotype has remained largely unexplored in soybean. In the present study, we indentified four Glycine max leaf yellow/green variegation mutants, Gmvar1, Gmvar2, Gmvar3, and Gmvar4, from artificial mutagenesis populations. Map-based cloning, together with the allelic identification test and CRISPR-based gene knockout, proved that mutated GmCS1 controls yellow-green variegation phenotype of the Gmvar mutants. GmCS1 encodes a chorismate synthase in soybean. The content of Phe, Tyr, and Trp were dramatically decreased in Gmcs1 mutants. Exogenous supply of three aromatic amino acid mixtures, or only Phe to Gmvar mutants, leads to recovery of the mutant phenotype. The various biological processes and signalling pathways related to metabolism and biosynthesis were altered in Gmvar mutants. Collectively, our findings provide new insights about the molecular regulatory network of yellow-green variegation leaf phenotype in soybean.

Accumulation of Polyphenolics and Differential Expression of Genes Related to Shikimate Pathway during Fruit Development and Maturation of Chinese Olive (Canarium album)

Phenolics in the Chinese olive (Canarium album (Lour.) Raeusch) fruit significantly affect its flavor and quality. The shikimate pathway is a bridge connecting primary metabolism and secondary metabolism through which fixed carbon can be transformed into phenolics. In this study, we aimed to reveal the relationship between the shikimate pathway and phenolic compound biosynthesis. Three Chinese olive fruits (cv. Tanxiang (TX), Changying (CY) and Lingfeng (LF)) with distinct flavor were utilized as materials. The results of this study showed that the synthesis and accumulation of quinate and gallate were active in the Chinese olive fruit. The accumulation amount of phenolic compounds was significantly different among the three cultivars. TX contained the highest content of ellagate, (iso)corilagin, conjugated quercetin and conjugated kaempferol; CY contained the highest content of conjugated luteolin; and LF contained the lowest content of ellagate, conjugated gallate, hyperin, conjugated quercetin, conjugated kaempferol and conjugated luteolin during fruit development. The expression of 3-dehydroquinate/shikimate dehydrogenase gene-4 (DHD/SDH-4), 3-dehydroquinate synthase gene (DHQS), chorismate synthase gene (CS) and Chorismate mutase gene-1 (CM-1) and shikimate content increased with the maturing of fruit. The gene 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase gene-1 (DAHPS-1) was most expressed in TX, while barely expressed in LF during fruit development. The expression of CM-1 was highest in CY. Chorismate mutase gene-2 (CM-2) expression was higher in TX and CY during late fruit development. The cultivars with higher expression of DAHPS-1 and Chorismate mutase genes (CMs) accumulated more phenolic compounds in fruit. DAHPS-1 and CMs are proposed as key genes for polyphenolic synthesis in the Chinese olive fruit. These results proved that shikimate metabolism had a positive effect on the phenols’ synthesis. Our study provides new insight into the regulatory mechanism of the biosynthesis and accumulation of phenolic compounds in the fruit of Chinese olive.

Subtractive sequence analysis aided druggable targets mining in Burkholderia cepacia complex and finding inhibitors through bioinformatics approach.

Burkholderia cepacia complex (BCC) is a group of gram-negative bacteria composed of at least 20 different species that cause diseases in plants, animals as well as humans (cystic fibrosis and airway infection). Here, we analyzed the proteomic data of 47 BCC strains by classifying them in three groups. Phylogenetic analyses were performed followed by individual core region identification for each group. Comparative analysis of the three individual core protein fractions resulted in 1766 ortholog/proteins. Non-human homologous proteins from the core region gave 1680 proteins. Essential protein analyses reduced the target list to 37 proteins, which were further compared to a closely related out-group, Burkholderia gladioli ATCC 10,248 strain, resulting in 21 proteins. 3D structure modeling, validation, and druggability step gave six targets that were subjected to further target prioritization parameters which ultimately resulted in two BCC targets. A library of 12,000 ZINC drug-like compounds was screened, where only the top hits were selected for docking orientations. These included ZINC01405842 (against Chorismate synthase aroC) and ZINC06055530 (against Bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/Glucosamine-1-phosphate acetyltransferase glmU). Finally, dynamics simulation (200ns) was performed for each ligand-receptor complex, followed by ADMET profiling. Of these targets, details of their applicability as drug targets have not yet been elucidated experimentally, hence making our predictions novel and it is suggested that further wet-lab experimentations should be conducted to test the identified BCC targets and ZINC scaffolds to inhibit them.

Multi‑locus sequence and drug resistance analysis of Salmonella infection in children with diarrhea in Guangdong to identify the dominant ST and cause of antibiotic‑resistance.

Multi-locus sequence typing (MLST) can be used to analyze the homology among the drug resistance gene cassettes in Salmonella and determine the prevalence. Information extracted using this technique can provide a theoretical basis for hospitals to devise protocols to control Salmonella infections. The aim of the present study was to investigate the possible association between drug resistance and integrons in clinical isolates of Salmonella from human fecal samples. Therefore, in the present study, 52 clinical fecal isolates of non-duplicate (i.e., not genome contamination) Salmonella were harvested from children with diarrhea and used for bacterial identification using biochemical tests, drug susceptibility analysis by antibiotic susceptibility testing and serotype identification using an agglutination assay. In total, seven Salmonella housekeeping genes (chorismate synthase, β sliding clamp of DNA polymerase III, uroporphyrinogen-III synthase, histidinol dehydrogenase, phosphoribosylaminoimidazole carboxylase catalytic subunit, 2-oxoglutarate dehydrogenase E1 component and homoserine dehydrogenase) were amplified and sequenced using MLST, before sequence alignment was performed against the Pub MLST database to determine the sequence-typed (ST) strains and construct genotypic evolutionary diagrams. Subsequently, the 52 Salmonella strains were subdivided into 11 serotypes and 11 sequence types. The dominant subtypes were found to be Salmonella typhimurium ST34 and ST19, which were diversely distributed. However, no new subtypes were found. Although the serotypes, including ST19, ST29, ST34, ST40, ST11, ST27, ST469, ST365, ST1499, ST413 and ST588, were closely associated with the MLST subtype, they did not correspond entirely. The detection rate of class I integrons was 38.46% (20/52), but no class II and III integrons were detected. The variable regions of three of 20 class I integrons were found to be amplified, whereas nine gene cassettes, including dihydrofolate reductase A12, open reading frame F, aminoglycoside-adenylyltransferase (aad)A2, aadA22, aadA23, aadA1, cadmium-translocating P-type ATPase 2, lincosamide and linF, were associated with drug resistance. These data suggest that Class I integrons are important factors underlying drug resistance in Salmonella, which may serve a role in the spread of drug resistance and warrant specific focus. In addition, MLST typing and serotyping should be applied cooperatively in epidemiological research.

In silico and In vitro Analysis of Nigella sativa Bioactives Against Chorismate Synthase of Listeria monocytogenes: a Target Protein for Biofilm Inhibition.

Listeria monocytogenes have the ability to form biofilms, which aid in the contamination of food and the evasion of antimicrobials. Consumption of L. monocytogenes laden food can promote mild to severe infection in humans and cause serious health issues. Therefore, biofilm development by L. monocytogenes is considered to be a major concern for both healthcare and food safety. This study attempted to target chorismate synthase, an essential protein predicted to be involved in the biofilm pathway. Nigella sativa is renowned for its applications in folk medicine; hence, bioactive ingredients reported were used for molecular docking studies. In the absence of a three-dimensional structure of chorismate synthase from L. monocytogenes, a homology model was generated using the Modeller program. A model with the highest DOPE score was chosen and validated. The reliable model was subjected to docking studies with 30 ligands from N. sativa. From this approach, α-longipinene was unveiled as the best hit. Further in vitro studies demonstrated the antibiofilm potential of α-longipinene against L. monocytogenes. Overall, the study reveals lead molecules from N. sativa as promising antibiofilm agents against L. monocytogenes. Hence, extended investigation with lead molecules will provide sustainable strategies to prevent biofilm-mediated problems due to L. monocytogenes.

De novo transcriptome analysis of Justicia adhatoda reveals candidate genes involved in major biosynthetic pathway.

BackgroundJusticia adhatoda is an important medicinal plant traditionally used in the Indian system of medicine and the absence of molecular-level studies in this plant hinders its wide use, hence the study was aimed to analyse the genes involved in its various pathways.Methods and resultsThe RNA isolated was subjected to Illumina sequencing. De novo assembly was performed using TRINITY software which produced 171,064 transcripts with 55,528 genes and N50 value of 2065 bp, followed by annotation of unigenes against NCBI, KEGG and Gene ontology databases resulted in 105,572 annotated unigenes and 40,288 non-annotated unigenes. A total of 5980 unigenes were mapped to 144 biochemical pathways, including the metabolism and biosynthesis pathways. The pathway analysis revealed the major transcripts involved in the tryptophan biosynthesis with TPM values of 6.0903, 33.6854, 11.527, 1.6959, and 8.1662 for Anthranilate synthase alpha, Anthranilate synthase beta, Arogenate/Prephenate dehydratase, Chorismate synthase and Chorismate mutase, respectively. The qRT-PCR validation of the key enzymes showed up-regulation in mid mature leaf when compared to root and young leaf tissue. A total of 16,154 SSRs were identified from the leaf transcriptome of J. Adhatoda ,which could be helpful in molecular breeding.ConclusionsThe study aimed at identifying transcripts involved in the tryptophan biosynthesis pathway for its medicinal properties, as it acts as a precursor to the acridone alkaloid biosynthesis with major key enzymes and their validation. This is the first study that reports transcriptome assembly and annotation of J. adhatoda plant.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11033-022-07784-5.

Physiological and Metabolic Response of Arthrospira maxima to Organophosphates.

The Spirulina spp. exhibited an ability to tolerate the organophosphates. This study aimed to explore the effects of the herbicide glyphosate on a selected strain of the cyanobacteria Arthrospira maxima cultivated in a company. Experimental cultivations acclimated in aquaria were treated with 0.2 mM glyphosate [N-(phosphonomethyl) glycine]. The culture biomass, the phycocyanin, and the chlorophyll a concentrations were evaluated every week during 42 days of treatment. The differentially expressed proteins in the treated cyanobacteria versus the control cultivations were evaluated weekly during 21 days of treatment. Even if the glyphosate treatment negatively affected the biomass and the photosynthetic pigments, it induced resistance in the survival A. maxima population. Proteins belonging to the response to osmotic stress and methylation pathways were strongly accumulated in treated cultivation; the response to toxic substances and the negative regulation of transcription seemed to have a role in the resistance. The glyphosate-affected enzyme, chorismate synthase, a key enzyme in the shikimic acid pathway, was accumulated during treatment, suggesting that the surviving strain of A. maxima expressed a glyphosate-resistant target enzyme.

Identification of potent inhibitors against chorismate synthase of Toxoplasma gondii using molecular dynamics simulations

Toxoplasmosis, caused by Toxoplasma gondii, affects about 20–30% of the human population every year globally. The emergence of severe side effects of current chemotherapeutics and drug-resistant strains emphasize upon finding new therapeutics to treat toxoplasmosis. Chorismate synthase (CS) is a vital enzyme of shikimate pathway and responsible for formation of chorismate, which acts as a precursor for production of several aromatic compounds important for virulence and survival in many bacteria and protozoans. In this study, comparative modeling was employed to predict the three-dimensional structure of T. gondii chorismate synthase (TgCS) followed by its refinement and validation using various computational tools. The modeled structure of TgCS monomer shows all the conserved features of CS, particularly the beta-alpha-beta sandwich fold. Molecular docking studies has displayed that 5-enolpyruvylshikimate-3-phosphate (EPSP, substrate) and flavin mononucleotide (FMN, cofactor) bind into the active site of TgCS and all the structures (apo, binary, and ternary) were observed to be stable during molecular dynamics (MD) simulation. Subsequently, structure-based virtual screening using TgCS has inferred two of each benzofuran and EPSP analogs as the best hits on the basis of RCS, molecular interactions, ADME properties, and MD simulations. The MD data of resultant protein–ligand complex structures was subjected to calculate the binding energy through MMPBSA method, which highlights that the EPSP analogs have higher binding affinity for the substrate-binding site of TgCS in comparison to benzofuran derivatives as well as substrate. Altogether, our study could pave the way for designing and development of next generation chemotherapeutic molecules against toxoplasmosis.

AroC, a chorismate synthase, is required for the formation of Edwardsiella tarda biofilms

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC, were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole.

Unraveling the possible inhibitors for Chorismate synthase to combat tuberculosis using in silico approach

Tuberculosis antibiotic resistance is a huge concern to the global population. The goal of this study was to find new and effective compounds to treat multidrug-resistant tuberculosis by targeting Chorismate synthase (CS), a crucial enzyme for Mycobacterium tuberculosis survival (MbT). The potential of a library of compounds as selective anti - tuberculosis drugs was investigated. Docking was first conducted using MoE to determine the effectiveness of the compounds. Molecular docking studies followed by MD simulation studies (total of 500 ns) in combination with free energy calculations grade the ligands in terms of their binding affinities. In the ligand bound state of the CS, MD simulations revealed a change from stretched to bended motional shift in loop L19. The RMSF analysis also revealed this flexibility, which was confirmed by visual inspection of L19 at various time intervals during the experiment. It appears that ZF1(-25.43Kcal/mol) and ZF2 (-22.04Kcal/mol) form hbonds and have a high binding energy in the active region of protein. Residues wise distribution of binding energy reveals that Arg144, Trp4, Thr6, and L19 amino acid residues are engaged in binding of CS with inhibitors. In summary, the findings suggest that compounds ZF1 and ZF2 may be more effective and selective anti-TB agents than currently available drugs. Also the role of L19, mediated by αH9 and αH5 in the retention of ligand inside the active pocket, through the formation of lid was also revealed. This knowledge will aid in the discovery of drugs that are potent CS inhibitors. More experimental research and a better understanding of the structure-activity relationship could aid in the development of possible candidates with better CS inhibition. Communicated by Ramaswamy H. Sarma

Growth inhibition of plasmodium falciparum by Nano-molar concentrations of 1-(4‑hydroxy-3-methoxyphenyl) decan-3-one (6-paradol); is a cure at hand?

BackgroundPlasmodium falciparum (P.f.)has developed resistance to most of the drugs that are being used currently. The continued search for a new drug is required to successfully eliminate the parasite. A pathway that is unique to the parasite is an ideal target to inhibit the growth of the parasite without affecting the host metabolism. PurposeIn this study, Zingiber officinale Roscoe (Zingiberaceae)(Z.o.) extract, used ethnobotanically as a potent antimicrobial, was found to inhibit P.f. in vitro. MethodsTo find the active compounds that may target the essential proteins of the P.f., the known secondary metabolite constituents of Z.o. rhizome were subjected to a preliminary in-silico target search. Chorismate synthase (CS) came up as a hit with a high binding score through multiple screening strategies. The antimalarial activity of the Z.o. extracts were tested with CS downstream metabolic products i.e. aromatic amino acids. The antimalarial targetablity of CS was confirmed with the phylogenetic conservancy, protein modeling, validation and subjected to High throughput virtual screening (HTVS) with a complete secondary metabolite library of Z.o. Among the top hits, the second hit 6-paradol (1-(4‑hydroxy-3-methoxyphenyl)decan-3-one) was available as a nature-identical synthetic compound. Compound 6-paradol was further analyzed for antimalarial and cytotoxic profiles and subjected to the same CS downstream product supplementation rescue assay. Results6-paradol is a highly potent antimalarial and a major component of Z.o. secondary metabolite pool. Though it is predicted to bind to the active site of chorismate synthase along with other metabolites of Z.o., an enzyme, part of the shikimate pathway involved in the aromatic amino acid synthesis. Parasite growth was rescued upon supplementation of aromatic amino acids along with crude Z.o. extract in parasite culture and a shift in IC50 of > 2 folds was observed, confirming the CS as the primary target of Z.o. crude extract. However, the rescue was inconsequential when we assayed with pure 6-paradol. ConclusionThis approach exploits natural compounds with high specificity and potency to kill pathogens developed due to eons of co-evolution with related pathogens. We report a novel potent antimalarial 6-paradol with nano-molar range IC50 already approved by FDA for human consumption as a nature-identical flavoring agent with immediate repurposing potential as an anti-malarial. However, CS rescue assay failure point to a different mechanism of action by active compounds and highlights Z.o. as a treasure trove of more potent anti-malarial which need further exploration.

Molecular Docking of Anti Helicobacter pylori Antibiotics and Proton Pump Inhibitor: A Single Center Survey

Helicobacter pylorus (H. pylori) is a deadly bacterium responsible for significant worldwide Gastric Cancer (GC) related mortality. The present study aimed to screen all the anti-microbial drugs used to eradicate H .pylori infection and to identify the most efficient drug by using computational methods through molecular docking analysis. The 3-D structure of protein chorismate synthase of H. pylori was downloaded from the Protein data bank (PDB) online browser. The x-ray crystallography structures of 13 common drugs used against H.pylori infection were also downloaded from the drug bank. We screened all 13 common drugs through molecular docking to know the most efficient binding interaction between the diverse ligand-protein complexes. The results were further compared with clinical survey data from the patients with diverse gastrointestinal H. pylori infected cases. Among the screened compounds, by in-silico approach we found that fluoroquinolone (FLRQ) and tetracycline (TET) manifested more significant interactions with chorismate synthase (CS) protein along with binding energies of -9.2 and -8.1 kcal/mole respectively. Further, the drugs were also corroborated with the survey data from patients with varied gastrointestinal disorders in our study. With this computational study, we could find FLRQ and TET may be the most efficient drug for H. pylori treatment, which can be tried in case of anti H. Pylori treatment failure due to resistance. Hence, effective inter-analysis between the experimental and computational approaches is crucial to build up a strong inhibitor.

Key aromatic amino acid players in soybean (Glycine max) genome under drought and salt stresses

In this study chorismate synthase, chorismate mutase and anthranilate synthase in the soybean genome (GmCS, GmCM, and GmAS or collectively named as GmAAAs in this study), were investigated using bioinformatics and expression analyses under salt and drought stresses. It is found that there are multiple copies of GmCM, GmCS, and GmAS in the soybean genome due to dispersed duplication; therefore, dispersion of paralog GmAAAs may indicate functional diversification of GmAAAs in the soybean genome. GmCM1, GmAS2 and GmAS3 genes were expressed above 1.5-fold in the tolerant cultivar according to results of digital expression analyses, suggesting that these three genes in drought-tolerant variety may be part of the drought tolerance mechanism rendering the cultivar tolerant to drought. The co-expression analyses indicate that some of the drought-regulated genes participate in the salinity stress response mechanism as well. The foliar gene expression analyses of selected GmAAAs showed that the highest gene expression under salt stress was in GmCM5 with 1.79-fold at 50 and 200 mM NaCl concentrations. Similarly, the highest upregulation of GmAAAs under drought stress was in GmCM5 with 2.23-fold. Generally, drought stress induced higher expressions of GmAAAs compared to salt stress in this study. Although the expression of aromatic amino acid genes differs depending on the duration of exposure to stress, plant organs and the type of abiotic stress; CM proteins may be one of the key players under both salt and drought stresses when the importance of AAA genes is considered. By and large, the findings presented in this study will enlighten the AAA pathway in plants, particularly in soybean.

Multifunctional Enzymes as Targets for the Treatment of Tuberculosis: Paving the Way for New Anti-TB Drugs.

In spite of the medical and technological developments of the last centuries, Tuberculosis (TB) has remained a challenging disease, with a limited number of therapeutic options, particularly in light of the increase in drug-resistant cases. The search for new molecules continues, with several candidates currently in clinical testing and ongoing efforts to identify novel targets. This work summarizes recent developments on anti-TB therapy, starting by discussing the current epidemiologic status and presenting an overview of the history of anti-tuberculosis drug discovery. Special attention is dedicated to five multifunctional enzymes that are regarded as promising targets for new anti-TB drugs: 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC); 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS)/GTP cyclohydrolase II (GCHII); glutamine dependent NAD+ Synthetase (NadE); chorismate synthase (CS); and Tryptophan synthase (TS). These enzymes are involved in metabolic pathways critical for the M. tuberculosis survival, growth or replication, but that are not expressed in humans or have significant differences in terms of functionality, which makes them appealing targets. Their function, structure, possible catalytic mechanisms, and current inhibition strategies and inhibitors are reviewed and discussed.

Chorismate synthase mediates cerebral malaria pathogenesis by eliciting salicylic acid-dependent autophagy response in parasite.

ABSTRACTCerebral malaria caused by Plasmodium falciparum is the severest form of the disease resulting in the morbidity of a huge number of people worldwide. Development of effective curatives is essential in order to overcome the fatality of cerebral malaria. Earlier studies have shown the presence of salicylic acid (SA) in malaria parasite P. falciparum, which plays a critical role in the manifestation of cerebral malaria. Further, the application of SA for the treatment of acute symptoms in cerebral malaria increases the activity of iNOS leading to severe inflammation-mediated death, also called as Reye's syndrome. Therefore, modulation of the level of SA might be a novel approach to neutralize the symptoms of cerebral malaria. The probable source of parasite SA is the shikimate pathway, which produces chorismate, a precursor to aromatic amino acids and other secondary metabolites like SA in the parasite. In this work, we performed the immunological, pathological and biochemical studies in mice infected with chorismate synthase knocked-out Plasmodium berghei ANKA, which does not produce SA. Fewer cerebral outcomes were observed as compared to the mice infected with wild-type parasite. The possible mechanism behind this protective effect might be the hindrance of SA-mediated induction of autophagy in the parasite, which helps in its survival in the stressed condition of brain microvasculature during cerebral malaria. The absence of SA leading to reduced parasite load along with the reduced pathological symptoms contributes to less fatality outcome by cerebral malaria.

Biophysical and In-Silico Studies of Phytochemicals Targeting Chorismate Synthase from Drug-Resistant Moraxella Catarrhalis

Chorismate serves as a crucial precursor for the synthesis of many aromatic compounds essential for the survival and virulence in various bacteria and protozoans. Chorismate synthase, a vital enzyme in the shikimate pathway, is responsible for the formation of chorismate from enolpyruvylshikimate-3-phosphate (EPSP). Moraxella catarrhalis is reported to be resistant to many beta-lactam antibiotics and causes chronic ailments such as otitis media, sinusitis, laryngitis, and bronchopulmonary infections. Here, we have cloned the aroC gene from Moraxella catarrhalis in pET28c and heterologously produced the chorismate synthase (~ 43kDa) in Escherichia coli BL21(DE3) cells. We have predicted the three-dimensional structure of this enzyme and used the refined model for ligand-based virtual screening against Supernatural Database using PyRx tool that led to the identification of the top three molecules (caffeic acid, gallic acid, and o-coumaric acid). The resultant protein-ligand complex structures were subjected to 50ns molecular dynamics (MD) simulation using GROMACS. Further, the binding energy was calculated by MM/PBSA approach using the trajectory obtained from MD simulation. The binding affinities of these compounds were validated with ITC experiments, which suggest that gallic acid has the highest binding affinity amongst these three phytochemicals. Together, these results pave the way for the use of these phytochemicals as potential anti-bacterial compounds.