Cysteine Synthase Research Articles

Lupeol Acetate and α-Amyrin Terpenes Activity against Trypanosoma cruzi: Insights into Toxicity and Potential Mechanisms of Action.

Chagas disease is a potentially fatal disease caused by the parasite Trypanosoma cruzi. There is growing scientific interest in finding new and better therapeutic alternatives for this disease's treatment. A total of 81 terpene compounds with potential trypanocidal activity were screened and found to have potential T. cruzi cysteine synthase (TcCS) inhibition using molecular docking, molecular dynamics, ADME and PAIN property analyses and in vitro susceptibility assays. Molecular docking analyses revealed energy ranges from -10.5 to -4.9 kcal/mol in the 81 tested compounds, where pentacyclic triterpenes were the best. Six compounds were selected to assess the stability of the TcCS-ligand complexes, of which lupeol acetate (ACLUPE) and α-amyrin (AMIR) exhibited the highest stability during 200 ns of molecular dynamics analysis. Such stability was primarily due to their hydrophobic interactions with the amino acids located in the enzyme's active site. In addition, ACLUPE and AMIR exhibited lipophilic characteristics, low intestinal absorption and no structural interferences or toxicity. Finally, selective index for ACLUPE was >5.94, with moderate potency in the trypomastigote stage (EC50 = 15.82 ± 3.7 μg/mL). AMIR's selective index was >9.36 and it was moderately potent in the amastigote stage (IC50 = 9.08 ± 23.85 μg/mL). The present study proposes a rational approach for exploring lupeol acetate and α-amyrin terpene compounds to design new drugs candidates for Chagas disease.

Dynamic association of the plastid localized cysteine synthase complex is vital for efficient cysteine production, photosynthesis, and grana thylakoid formation in transgenic tobacco.

Cysteine biosynthesis is essential for translation and represents the entry point of reduced sulfur into plant metabolism. The two consecutively acting enzymes serine-acetyltransferase (SAT) and O-acetylserine-thiol-lyase catalyze cysteine production and form the cysteine synthase complex (CSC), in which SAT is activated. Here we show that expression of active SAT in plastids of tobacco (PSA) causes substantial cysteine accumulation. Remarkably, enhanced cysteine production in plastids entirely abolished grana stack formation, impaired photosynthesis capacity, and decreased the number of chloroplasts in mesophyll cells of PSA lines. Transgenic tobacco lines expressing active SAT in the cytosol (CSA) accumulated comparable amounts of thiols but displayed no phenotype. To dissect the consequences of CSC formation from enhanced SAT activity in tobacco plastids, we expressed an enzymatically inactive SAT that can still form the CSC in tobacco plastids (PSI). The PSI lines were indistinguishable from PSA lines, although the PSI lines displayed no increase in plastid-localized SAT activity. Neither PSA lines nor PSI lines suffered from an oxidized redox environment in plastids that could have been causative for the disturbed photosynthesis. From these findings, we infer that the association of the plastid CSC itself triggers a signaling cascade controlling sulfur assimilation and photosynthetic capacity in leaves.

Cysteine Biosynthesis in Campylobacter jejuni: Substrate Specificity of CysM and the Dualism of Sulfide.

Campylobacter jejuni is a highly successful enteric pathogen with a small, host-adapted genome (1.64 Mbp, ~1650 coding genes). As a result, C. jejuni has limited capacity in numerous metabolic pathways, including sulfur metabolism. Unable to utilise ionic sulfur, C. jejuni relies on the uptake of exogenous cysteine and its derivatives for its supply of this essential amino acid. Cysteine can also be synthesized de novo by the sole cysteine synthase, CysM. In this study, we explored the substrate specificity of purified C. jejuni CysM and define it as an O-acetyl-L-serine sulfhydrylase with an almost absolute preference for sulfide as sulfur donor. Sulfide is produced in abundance in the intestinal niche C. jejuni colonises, yet sulfide is generally viewed as highly toxic to bacteria. We conducted a series of growth experiments in sulfur-limited media and demonstrate that sulfide is an excellent sulfur source for C. jejuni at physiologically relevant concentrations, combating the view of sulfide as a purely deleterious compound to bacteria. Nonetheless, C. jejuni is indeed inhibited by elevated concentrations of sulfide and we sought to understand the targets involved. Surprisingly, we found that inactivation of the sulfide-sensitive primary terminal oxidase, the cbb3-type cytochrome c oxidase CcoNOPQ, did not explain the majority of growth inhibition by sulfide. Therefore, further work is required to reveal the cellular targets responsible for sulfide toxicity in C. jejuni.

Insights into the loss of glucoraphanin in post-harvested broccoli––Possible involvement of the declined supply capacity of sulfur donor

The loss of characteristic nutrient glucoraphanin during the shelf life seriously affects the nutritional quality of broccoli. Here, we monitored the changes in the levels of sulfur donors (cysteine and glutathione) required for glucoraphanin biosynthesis. Similar to glucoraphanin, cysteine content decreased sharply. Continuous down-regulation of BoCysK1 and BoCysK2 genes encoding cysteine synthase might account for cysteine loss. Contrarily, glutathione content accumulated steadily, which might owe to the up-regulation of biosynthetic gene (BoEC1). Additionally, the change of malondialdehyde content was positively correlated with glutathione, implying that oxidative stress might stimulate glutathione accumulation. Nevertheless, the expression of BoGSTF11 gene encoding glutathione S-transferases was down-regulated, which blocked the supply of glutathione. The increase in the content of raphanusamic acid (degradation product) indicated that insufficient supply of sulfur donors not only could constrain the biosynthesis of glucoraphanin but also triggered its degradation.

A comparative study on antibiotic resistance and virulence properties of Staphylococcus aureus isolated from hospitalized patients and hospital environment

We compared the characteristics of clinical Staphylococcus aureus and S. aureus isolated from environmental surfaces in 3 hospitals. Clinical S. aureus isolates were collected from hospitalized patients. Environmental surfaces were sampled from the rooms of patients infected with S. aureus. After identifying rooms with the target organism, 3-5 high-touch surfaces in patient care areas were sampled using swabs before room cleaning by environmental services. S. aureus isolates were subjected to genotyping, antimicrobial susceptibility testing, and virulence determinant screening. The isolates were analyzed for integron content and sequences of variable region amplification products. There were epidemiologically unrelated 79 clinical and 62 environmental S. aureus isolates. Overall, 11.4% of clinical and 59.7% of environmental isolates were methicillin-resistant. The environmental and clinical S. aureus exhibited very different virulence profiles: 79% of the environmental isolates were negative for virulence genes compared to 2.5% of clinical isolates (P < .001). Environmental isolates were more resistant to antibiotics compared to clinical isolates. Class 1 integrons were only detected in 7 of 62 environmental isolates, of which 3 isolates had integrons with cysteine synthase cassette, 1 had aadA1, and 1 had an unknown cassette. These data indicate the different characteristics between environmental and clinical S. aureus, which may reflect different reservoirs from which the 2 groups acquired the strains.

Sodium hydrosulfide alleviates aluminum toxicity in Brassica napus through maintaining H2S, ROS homeostasis and enhancing aluminum exclusion

Hydrogen sulfide (H2S) is a gaseous mediator that plays versatile roles in plant growth and stress responses. However, the regulatory functions of H2S in plant responses to aluminum (Al) stress remain elusive. We observed that application of 20 μM of NaHS (H2S donor) or 0.2 mM of hypotaurine (HT, H2S scavenger) significantly mitigated the inhibition of rapeseed root growth caused by Al stress (150 μM). Exposure to Al for 6 h induced significant H2S accumulation and high levels were maintained thereafter, owing to the elevation of cysteine (83.73 %), L-cysteine desulfhydrase (LCD, 92.32 %), and cyanoalanine synthase (CAS, 11.23 %), and the inhibition of O-Acetyl-l-serine (thiol) lyase (OAS-TL, 15.13 %). Addition of HT significantly scavenged the prolonged H2S accumulation caused by Al stress. Exogenous NaHS maintained the H2S homeostasis through increasing OAS-TL activity (34.99 %) and inhibiting LCD activity (25.72 %), and cysteine level (39.53 %). Moreover, exogenous NaHS mitigated oxidative damage by enhancing antioxidant enzyme activity (SOD 26.27 %, POD 28.62 %, CAT 400.5 % and APX 92.68 %) and proline content (19.85 %). It also decreased root cell wall Al accumulation (20.52 %) by decreasing PME activity (24.64 %) and facilitating pectin methylation (16.74 %). Similar alleviative effects were observed when HT was added. These results suggest that H2S functions differential roles in Al stress response in rapeseed seedlings, depending on its local concentration and duration. Prolonged high H2S emissions might contribute to Al toxicity, while moderate exogenous H2S improves Al tolerance through controlling H2S and ROS accumulation and enhancing Al exclusion through replenishing antioxidant reservoirs and facilitating pectin methylation. It is therefore important that further study investigates how to orchestrate endogenous H2S levels and improve plant stress tolerance.

Starch accumulation dynamics and transcriptome analysis of Chlorella sorokiniana during transition of sulfur nutritional status

Microalgae can effectively accumulate starch by using nutritional limitation methods in the context of bioalcohol fuel production. However, relatively few studies have focused on starch accumulation in microalgae and its molecular basis, especially under sulfur limitation conditions. In this study, the starch accumulation dynamics and physiological responses of Chlorella sorokiniana under sulfur starvation (SS) and sulfur replenishment (SR) conditions were investigated, and the genes involved in the transcriptional regulation were explored using RNA-seq. The starch content in C. sorokiniana cells significantly increased from 1.6% to 55.0% of dry weight within 24 h under SS conditions, and then, it decreased to 3.4% within 12 h after transition to SR conditions. However, cell growth was inhibited, and pigment content decreased under SS conditions. Using RNA-seq analysis, a total of 9720 differentially expressed genes (DEGs) induced by sulfur status were obtained. These genes were narrowed down to 454 starvation and replenishment cross-validated (SRV)-DEGs, among which 283 SRV-DEGs were significantly up-regulated and 171 SRV-DEGs were down-regulated under SS conditions, and returned to their previous state under SR conditions. The SRV-DEGs enriched in the sulfate metabolism pathway were all up-regulated under SS conditions after 6 h to speed up the sulfur metabolic cycle, and the transcriptional abundance of a sulfate transporter (SULTR4), cysteine synthase[O-acetylserine(thiol)-lyase] (OASTL), serine acetyltransferase (SAT), and methanethiol oxidase (SELENBP1) increased 8.6-fold, 12.6-fold, 8.7-fold, and 12.4-fold, respectively. Protein synthesis was correspondingly inhibited, which resulted in the reallocation of carbon and elevated the starch synthesis pathway, in which the expressions of glycogen branching enzyme (GBE) and starch synthase (SS) were up-regulated 12.0- and 3.0-fold, respectively. The fatty acid desaturase (FAD) and phosphatidic acid phosphatase (PAP) in the lipid synthesis pathway were strongly up-regulated 8.8- and 16.2-fold, respectively, indicating the competitive synthesis of lipids. The down-regulation of SRV-DEGs associated with carbon fixation, such as those in the Calvin cycle, possibly affected cell growth. The time-resolved transcriptional analysis identified the SRV-DEGs, revealing the underlying starch accumulation mechanism, as well as the relationship with cell growth and lipid synthesis.

Cysteine synthase: a key enzyme in the cysteine synthesis pathway and a novel drug target for Chagas disease

Cysteine synthase: a key enzyme in the cysteine synthesis pathway and a novel drug target for Chagas disease

Alfalfa’s response to atrazine stress and its secreted atrazine metabolites

Although listed as endocrine disruptor compounds, atrazine (ATZ) is still used in large quantities in agricultural production. Here, alfalfa seedling was cultivated in hydroponic media to investigate the toxic effects of ATZ on alfalfa and accumulation of ATZ in tissues of different plant parts. Alfalfa had a strong upward translocation ability to ATZ. The stress response of alfalfa under ATZ stress was studied using metabolomic and transcriptomic techniques. S-adenosylmethionine, glutathione, 3-mercaptopyruvic acid, ornithine, and aminopropylcadaverine were significantly increased by ATZ in pathways mtr00270 and mtr00480. Several genes of cysteine synthase and spermidine synthase were significantly up-regulated by ATZ induction. They may be markers and genes with potential physiological functions of alfalfa in response to ATZ stress. In addition, using high resolution mass spectrometry, a total of five ATZ metabolites secreted from alfalfa roots were detected. Among them, acetylated deisopropylated ATZ was discovered for the first time. Hydroxylated ATZ and acetylated deethylated ATZ were more readily excreted by the root system. This study not only provides potential genes for the construction of engineering plants to remediate ATZ-contaminated soil, but also provides monitoring objects for the ecological research of ATZ metabolites.

A novel approach using protein-rich biomass as co-fermentation substrates to enhance phosphorus recovery from FePs-bearing sludge

A novel approach for the enhancement of phosphorus (P) recovery from Fe bound P compounds (FePs)-bearing sludge by co-fermentation with protein-rich biomass (PRB) is reported. Four PRBs (silkworm chrysalis meal, fish meal, corn gluten meal, and soya bean meal) were used for co-fermentation. The results revealed that PRBs with strong surface hydrophobicity and loose structure favored the hydrolysis and acidogenesis processes. Sulfide produced by PRB could react with FePs to form FeS and promote P release. Due to the neutralization of volatile fatty acids (VFAs) by a relatively high concentration of ammonia, the pH was maintained near neutral and thus prevented the dissolution of metal ions (e.g., Fe and Ca). This was beneficial to save the cost of subsequent P recovery and form high-purity struvite. Compared with the control, the soluble orthophosphate and VFAs increased by 88.3% and 531.3%, respectively, in the co-fermentation system with silkworm chrysalis meal. Cysteine was the important intermediate. The metagenomics analysis indicated that the gene abundances of phosphate acetyltransferase and acetate kinase, which were key enzymes in the acetate metabolism, increased by 117.7% and 52.2%, respectively. The gene abundances of serine O-acetyltransferase and cysteine synthase increased by 63.4% and 54.4%, respectively. Cysteine was primarily transformed to pyruvate and sulfide. This study provides an environment-friendly strategy to simultaneously recover P and VFAs resources from FePs-bearing sludge and PRB waste.

β-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified β-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and β-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its β-cyanoalanine synthase activity. Consistent with the β-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the β-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite’s ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

Synthesis and Structure-Activity Relationship of Thioacetamide-Triazoles against Escherichia coli.

Infections due to Gram-negative bacteria are increasingly dangerous due to the spread of multi-drug resistant strains, emphasizing the urgent need for new antibiotics with alternative modes of action. We have previously identified a novel class of antibacterial agents, thioacetamide-triazoles, using an antifolate targeted screen and determined their mode of action which is dependent on activation by cysteine synthase A. Herein, we report a detailed examination of the anti-E. coli structure–activity relationship of the thioacetamide-triazoles. Analogs of the initial hit compounds were synthesized to study the contribution of the aryl, thioacetamide, and triazole sections. A clear structure–activity relationship was observed generating compounds with excellent inhibition values. Substitutions to the aryl ring were generally best tolerated, including the introduction of thiazole and pyridine heteroaryl systems. Substitutions to the central thioacetamide linker section were more nuanced; the introduction of a methyl branch to the thioacetamide linker substantially decreased antibacterial activity, but the isomeric propionamide and N-benzamide systems retained activity. Changes to the triazole portion of the molecule dramatically decreased the antibacterial activity, further indicating that 1,2,3-triazole is critical for potency. From these studies, we have identified new lead compounds with desirable in-vitro ADME properties and in-vivo pharmacokinetic properties.

Serine acetyltransferase from Neisseria gonorrhoeae; structural and biochemical basis of inhibition

Serine acetyltransferase (SAT) catalyzes the first step in the two-step pathway to synthesize l-cysteine in bacteria and plants. SAT synthesizes O-acetylserine from substrates l-serine and acetyl coenzyme A and is a key enzyme for regulating cellular cysteine levels by feedback inhibition of l-cysteine, and its involvement in the cysteine synthase complex. We have performed extensive structural and kinetic characterization of the SAT enzyme from the antibiotic-resistant pathogen Neisseria gonorrhoeae. Using X-ray crystallography, we have solved the structures of NgSAT with the non-natural ligand, l-malate (present in the crystallization screen) to 2.01 Å and with the natural substrate l-serine (2.80 Å) bound. Both structures are hexamers, with each monomer displaying the characteristic left-handed parallel β-helix domain of the acyltransferase superfamily of enzymes. Each structure displays both extended and closed conformations of the C-terminal tail. l-malate bound in the active site results in an interesting mix of open and closed active site conformations, exhibiting a structural change mimicking the conformation of cysteine (inhibitor) bound structures from other organisms. Kinetic characterization shows competitive inhibition of l-cysteine with substrates l-serine and acetyl coenzyme A. The SAT reaction represents a key point for the regulation of cysteine biosynthesis and controlling cellular sulfur due to feedback inhibition by l-cysteine and formation of the cysteine synthase complex. Data presented here provide the structural and mechanistic basis for inhibitor design and given this enzyme is not present in humans could be explored to combat the rise of extensively antimicrobial resistant N. gonorrhoeae.

Genome-Wide Investigation of the Cysteine Synthase Gene Family Shows That Overexpression of CSase Confers Alkali Tolerance to Alfalfa (Medicago sativa L.).

Alfalfa is widely grown worldwide as a perennial high-quality legume forage and as a good ecological landcover. The cysteine synthase (CSase) gene family is actively involved in plant growth and development and abiotic stress resistance but has not been systematically investigated in alfalfa. We identified 39 MsCSase genes on 4 chromosomes of the alfalfa genome. Phylogenetic analysis demonstrated that these genes were clustered into six subfamilies, and members of the same subfamily had similar physicochemical properties and sequence structures. Overexpression of the CSase gene in alfalfa increased alkali tolerance. Compared with control plants, the overexpression lines presented higher proline, soluble sugars, and cysteine and reduced glutathione contents and superoxide dismutase and peroxidase activities as well as lower hydrogen peroxide and superoxide anion contents after alkali stress. The relative expression of γ-glutamyl cysteine synthetase gene (a downstream gene of CSase) in the overexpression lines was much higher than that in the control line. The CSase gene enhanced alkalinity tolerance by regulating osmoregulatory substances and improving antioxidant capacity. These results provide a reference for studying the CSase gene family in alfalfa and expanding the alkali tolerance gene resources of forage plants.

Microbial consortium mediated growth promotion and Arsenic reduction in Rice: An integrated transcriptome and proteome profiling

The adverse effects of arsenic (As) contamination are well known. Rice is a staple food for 50% of the world population but the accumulation of As into rice hampers the food security and safety. Thus the amelioration of As stress and reduction of As levels in rice are needed. In this study, transcriptome (Illumina sequencing) and proteome (2D gel electrophoresis) explored mechanisms of consortium (P. putida+C. vulgaris) mediated growth promotion and As amelioration in rice. The rice seedlings grown hydroponically in the Hewitt nutrient medium and after acclimatization, exposed to 50 µM As alone as well as with microbial consortium to observe the impact at morphological and molecular level. The inoculation of microbial consortium significantly ameliorated the As toxicity, improved growth of root hairs and maintained cellular integrity of the epidermis, exodermis and the stele region during As exposure. Several genes showed differential expression in As and As+P. putida. Down-regulation of As transporters (OsPIP2;2 and OsPIP2;3, OsTIP2;1) and higher expression of WRKY gene (WRKY28) during As+P. putida+C.vulgaris suggested reduction of As uptake in rice. The up-regulation of nutrient elements transporters (OsZIFL9, OsZIFL5, OsZIFL12 and OsZIP2, OsYSL15 and OsCOPT6) in the presence of consortium indicated the improved nutrient status of rice. Higher expression of regulatory elements like auxin/indole 3 acetic acid (AUX/IAA), WRKY and myeloblastosis (MYB) TFs and down-regulation of defense responsive genes such Glutathione-S-transferase, Peroxidase and Glutaredoxinduring As+P. putida+C.vulgaris exposure was also observed. Proteome profiling demonstrated differential abundance of proteins involved in photosynthesis (chlorophyll a/b binding protein, photosystem I Fe-S centre), energy metabolism (ATP synthase subunit beta) transport, signaling (tubulin 1, actin 1), defense (glutathione S-transferase, phenylalanine ammonia lyase) and amino acid metabolism (cysteine synthase, glutamine synthetase), which supported the As ameliorative and growth-promoting potential of microbial consortium during As stress in rice plants. The study gives comprehensive information about gene and protein changes in rice plants in As+consortium exposure.

Transcriptome and proteome profiling revealed molecular mechanism of selenium responses in bread wheat (Triticum aestivum L.)

BackgroundAlthough selenium (Se) plays important roles in scavenging free radicals, alleviating oxidative stresses, and strengthening immune system, the knowledge about Se responses in bread wheat is still limited. In order to clarify the molecular mechanism of Se responses in bread wheat, 2-week-old wheat seedlings of cultivar ‘Jimai22’ treated with 10 μM disodium selenate (Na2SeO4) for 0, 3, and 24 h were collected and analyzed by transcriptional sequencing and tandem mass tag-based (TMT) quantitative proteomics.ResultsAt least 11,656 proteins and 133,911 genes were identified, and proteins including ATP sulfurylase (APS), cysteine synthase (CS), SeCys lyase, sulfate transporters, glutathione S-transferase (GSTs), glutathione peroxidase (GSH-Px), glutaredoxins (GRXs), superoxide dismutases (SODs), catalases (CATs), heat shock proteins (HSPs), UDP-glycose flavonoid glycosyltransferases (UFGTs), sucrose-6-phosphate hydrolases (Suc-6-PHs), archaeal phosphoglucose isomerases (APGIs), malate synthases (MSs), and endo-1,4-beta-xylanase (Xyn) in Se accumulation, ROS scavenging, secondary metabolism, and carbohydrate metabolism were significantly differently expressed.ConclusionsThis is the first complementary analyses of the transcriptome and proteome related with selenium responses in bread wheat. Our work enhances the understanding about the molecular mechanism of selenium responses in bread wheat.

Sulfide-quinone oxidoreductase is required for cysteine synthesis and indispensable to mitochondrial health

Mitochondrial dysfunction is related to common age-related disorders, including neurodegenerative diseases, metabolic syndrome, and carcinogenesis. Therefore, maintaining the functionality and integrity of mitochondria is important for human health. Herein, we found that sulfide:quinone oxidoreductase (Sqr), which oxidizes hydrogen sulfide to reactive sulfur species (RSS), was indispensable to mitochondria health in the eukaryotic model microorganism Schizosaccharomyces pombe. Sqr knock-out led to morphological changes and functional deficiencies of mitochondria and apoptosis in S. pombe. The Sqr knock-out strain displayed the same phenotypes as the cysteine-synthesis-deficient strain, and cysteine addition complemented the effects caused by Sqr knock-out. In S. pombe, Sqr was the main RSS producer in mitochondria, and RSS instead of H2S was used by cysteine synthase to synthesize cysteine. This finding rewrites the cysteine biosynthesis route in S. pombe and may also in other eukaryotes and prokaryotes, and highlights the importance of cysteine and RSS in maintaining mitochondrial health.

Prediction of potential cysteine synthase inhibitors of Leishmania braziliensis and Leishmania major parasites by computational screening

Leishmaniasis is a neglected tropical disease considered a public health problem that requires innovative strategies for its chemotherapeutic control. In the present investigation, a molecular docking approach was carried out using the protein cysteine synthase (CS) of Leishmania braziliensis (CSLb) and Leishmania major (CSLm) parasites to identify new compounds as potential candidates for the development of selective leishmaniasis therapy. CS protein sequence similarity, active site, structural modeling, molecular docking, and ADMET properties of compounds were analyzed using bioinformatics tools. Molecular docking analyses identified 1000 ligands with highly promising binding affinity scores for both CS proteins. A total of 182 compounds for CSLb and 173 for CSLm were selected for more detailed characterization based on the binding energy and frequency values and ADMET properties. Based on Principal Component Analysis (PCA) and K-means clusterization for both CS proteins, we classified compounds into 5 clusters for CSLb and 7 for CSLm, thus providing an excellent starting point for verification of enzyme inhibition in in vitro studies. We found the ZINC16524774 compound predicted to have a high affinity and stability for both CSLb and CSLm proteins, which was also evaluated through molecular dynamics simulations. Compounds within each of the five clusters also displayed pharmacological and structural properties that make them attractive drug candidates for the development of selective cutaneous leishmaniasis chemotherapy.

Moonlighting Biochemistry of Cysteine Synthase: A Species-specific Global Regulator

Cysteine Synthase (CS), the enzyme that synthesizes cysteine, performs non-canonical regulatory roles by binding and modulating functions of disparate proteins. Beyond its role in catalysis and regulation in the cysteine biosynthesis pathway, it exerts its moonlighting effect by binding to few other proteins which possess a C-terminal “CS-binding motif”, ending with a terminal ILE. Therefore, we hypothesized that CS might regulate many other disparate proteins with the “CS-binding motif”. In this study, we developed an iterative sequence matching method for mapping moonlighting biochemistry of CS and validated our prediction by analytical and structural approaches. Using a minimal protein-peptide interaction system, we show that five previously unknown CS-binder proteins that participate in diverse metabolic processes interact with CS in a species-specific manner. Furthermore, results show that signatures of protein–protein interactions, including thermodynamic, competitive-inhibition, and structural features, highly match the known CS-Binder, serine acetyltransferase (SAT). Together, the results presented in this study allow us to map the extreme multifunctional space (EMS) of CS and reveal the biochemistry of moonlighting space, a subset of EMS. We believe that the integrated computational and experimental workflow developed here could be further modified and extended to study protein-specific moonlighting properties of multifunctional proteins.

Screening and identification of salt-tolerance genes in Sophora alopecuroides and functional verification of SaAQP.

Overexpression of SaAQP can improve the salt tolerance of transgenic soybean hairy roots and A. thaliana. Salt stress severely affects crop yield and food security. There is a need to improve the salt tolerance of crops, but the discovery and utilization of salt-tolerance genes remains limited. Owing to its strong stress tolerance, Sophora alopecuroides is ideal for the identification of salt-tolerance genes. Therefore, we aimed to screen and identify the salt-tolerance genes in S. alopecuroides. With a yeast expression library of seedlings, salt-tolerant genes were screened using a salt-containing medium to simulate salt stress. By combining salt-treatment screening and transcriptome sequencing, 11 candidate genes related to salt tolerance were identified, including genes for peroxidase, inositol methyltransferase, aquaporin, cysteine synthase, pectinesterase, and WRKY. The expression dynamics of candidate genes were analyzed after salt treatment of S. alopecuroides, and salt tolerance was verified in yeast BY4743. The candidate genes participated in the salt-stress response in S. alopecuroides, and their overexpression significantly improved the salt tolerance of yeast. Salt tolerance mediated by SaAQP was further verified in soybean hairy roots and Arabidopsis thaliana, and it was found that SaAQP might enhance the salt tolerance of A. thaliana by participating in a reactive oxygen species scavenging mechanism. This result provides new genetic resources in plant breeding for salt resistance.