Serine Acetyltransferase Research Articles

Transcriptome analysis of grape leaves reveals insights into response to heat acclimation

Plants subjected to mild heat stress can acquire thermotolerance. But little is known about the how a grape changes, at the transcription level, after heat acclimation. In this study, we compared the transcriptome data of grape leaves after heat treatment between heat-acclimatized and non-heat-acclimatized plants, in order to detect the molecular mechanism of the acquired thermotolerance. Heat acclimation significantly relieves the heat injury of grape, with lower relative electrolyte leakage (REL) and higher Fv/Fm in heat-acclimatized plants. A total of 1526 shared genes (854 up- and 672 down-regulated) were in heat treatment (H) and heat acclimation treatment (HA), and 3533 differently expressed genes (DEGs) were unique to HA treatment. By GO analysis, DEGs in H treatment were enriched in signal transduction pathways, while DEGs in HA treatment were enriched in macromolecule metabolic processes. Abscisic acid (ABA) signaling pathway was activated by H and HA treatment. Meanwhile, 25 heat shock proteins (HSPs), 11 antioxidase genes, and 31 transcription factor genes were up-regulated. Notably, heat acclimation suppressed sucrose metabolism pathway, and sucrose synthase (SUS) and sucrose phosphate synthase 3 (SPS3) were down-regulated. The nucleotide excision repair (NER) pathway was activated by HA treatment, and 8 genes in this pathway were induced. Heat acclimation also activated the cysteine biosynthesis pathway, in which serine acetyltransferase 1 (SAT1) and S-sulfocysteine synthase (SSCS) expressed at high levels. Transcriptomic profiling reveals transcriptional alterations in heat-acclimatized and non-heat-acclimatized grapevines under heat stress, which provides a new insight into the plant response to heat acclimation.

Sulfur metabolic engineering enhances cadmium stress tolerance and root to shoot iron translocation in Brassica napus L

Serine acetyltransferase (SAT) (EC 2.3.1.30) is the rate-limiting enzyme of cysteine (Cys) biosynthesis, providing the decisive precursor for the ubiquitous defense thiol glutathione (GSH). Together with O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) SAT generates Cys in the cytosol, plastids, and mitochondria of vascular plants. The current study aimed to overproduce Cys and GSH for enhanced stress tolerance via overexpression of the feedback-insensitive isoform of serine acetyltransferase from tobacco, i.e., NtSAT4. Constitutive overexpression of NtSAT4 in Brassica napus resulted in the 2.6-fold–4-fold higher SAT activity in different subcellular compartment-specific lines. This higher SAT activity led to a 2.5-fold–3.5-fold higher steady-state level of free Cys and 2.2-fold–5.3-fold elevated level of GSH in leaves compared with nontransformed plants. Among the compartment-specific lines, the mitochondrial targeted NtSAT4 overexpressor line M-182 showed the highest levels of Cys (3.5-fold) and GSH (5.3-fold) compared with wild-type plants. Overexpression of NtSAT4 conferred a physiological advantage in terms of enhanced tolerance against oxidative stress with hydrogen peroxide and the heavy metal cadmium (Cd). The NtSAT4 overexpressor lines showed a significantly higher amount of iron (Fe) translocation from roots to shoots compared with nontransformed plants. Overall, these results suggest that overexpression of NtSAT4 is a promising approach to creating plants with tolerance to heavy metals and oxidative stress and, in addition, may potentially improve plant nutrition in terms of enhanced Fe translocation from roots to shoots.

MRPA-independent mechanisms of antimony resistance in Leishmania infantum

Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum, and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA−/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA−/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of satQ390K, satG321R and satG325R variants led to a 2–3.2 -fold increase in Sb resistance in mrpA−/- parasites. Only satG321R and satG325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites.

Photosynthetic activity and RAPD profile of polyethylene glycol treated B. juncea L. under nitric oxide and abscisic acid application

The involvement of two extremely important signalling molecules, nitric oxide (NO) and abscisic acid (ABA) has been employed by plants to facilitate the adaptive/tolerate response during stressful conditions. However, the interactive role of exogenously applied NO and ABA is very less studied at physiological, biochemical and molecular levels. The present study therefore, evaluated the effects of individual and simultaneous addition of exogenous NO donor SNP (100μM) and ABA (10μM) on photosynthesis, Calvin-Benson cycle enzymes, S-assimilation enzymes, oxidative stress components, and genotoxicity in Brassica juncea cv. Varuna, exposed to polyethylene glycol (PEG)-induced drought stress. Results showed that a loss induced by PEG was significantly surpassed by the application of NO or/and ABA with PEG for chlorophyll content, net photosynthestic rate (Pn), internal CO2 concentration (Ci), stomatal conductance (gs), transpiration rate (Tr), maximum photosystem II (PSII) efficiency (Fv/Fm), actual PSII efficiency (ΦPSII), intrinsic PSII efficiency (Fv´/ Fm´), photochemical quenching (qP), non-photochemical quenching (NPQ), electron transport chain (ETC), ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo), glyceraldehyde-3-phosphate dehydrogenase (GapDH), phosphoribulokinase (PRK), ATP-sulfurylase (ATP-S), and serine acetyltransferase (SAT) activities. The genomic template stability (GTS) (measured as changes in RAPD profiles) was significantly affected and showed varying degrees of DNA polymorphism, highest in PEG and lowest in PEG + NO and PEG + NO + ABA. Furthermore, the changes in RAPD profiles showed consistent results when compared with various photosynthetic and oxidative parameters. Altogether, this study concluded that supplementation of individual NO and together with ABA was more effective than individual ABA in alleviating PEG-induced drought stress in B. juncea L. seedlings.

Copper accumulation and biochemical responses of Sesuvium portulacastrum (L.)

Sesuvium portulacastrum (L.) is a halophytic phytoremediator plant with potential for significant accumulation of metals and metalloids. The present work evaluatedthe physiological and biochemical responses of Sesuvium plants to copper (Cu) exposure (100–500 μM) for 30 d in field conditions. Plants demonstrated significant copper accumulation that increased with the increase in Cu concentration of the medium (maximum 254 µg g−1 DW at 500 µM). The root dry weight was not significantly affected at 500 μM while shoot dry weight decreased significantly. Total soluble proteins, photosynthetic pigmentsand malondialdehye (MDA) were declined significantly beyond 100 μM after 30 d. Among metabolites and enzymes of thiol metabolism, total non-protein thiols (NP-SH), γ-glutamylcysteine synthetase and glutathione reductase did not show significant effect while cysteine, serine acetyltransferase, and cysteine synthaseshowed a significant decline beyond 100 μM. The level of proline, glycine betaine and total phenolics also showed decreasing trend with the increase in Cu concentration. In conclusion, plantsare potential phytoextractor of Cu but do suffer from the toxic effects of Cu at high concentration of 500 μM. Sesuvium plants therefore appear suitable for use in phytoremediation purpose at low Cu concentrations (100 – 250 μM).

Molecular cloning of putative chloroplastic cysteine synthase in Leucaena leucocephala.

Cysteine biosynthesis is directed by the successive commitments of serine acetyltransferase, and O-acetylserine (thiol) lyase (OASTL) compounds, which subsequently frame the decameric cysteine synthase complex. The isoforms of OASTL are found in three compartments of the cell: the cytosol, plastid, and mitochondria. In this investigation, we first isolated putative chloroplastic OASTL (Ch-OASTL) from Leucaena leucocephala, and the Ch-OASTL was then expressed in BL21-competent Escherichia coli. The putative Ch-OASTL cDNA clone had 1,543 base pairs with 391 amino acids in its open reading frame and a molecular weight of 41.54kDa. The purified protein product exhibited cysteine synthesis ability, but not mimosine synthesis activity. However, they both make the common α-aminoacrylate intermediate in their first half reaction scheme with the conventional substrate O-acetyl serine (OAS). Hence, we considered putative Ch-OASTL a cysteine-specific enzyme. Kinetic studies demonstrated that the optimum pH for cysteine synthesis was 7.0, and the optimum temperature was 40°C. In the cysteine synthesis assay, the Km and kcat values were 838 ± 26µM and 72.83s-1 for OAS, respectively, and 60 ± 2µM and 2.43s-1 for Na2S, respectively. We can infer that putative Ch-OASTL regulatory role is considered a sensor for sulfur constraint conditions, and it acts as a forerunner of various metabolic compound molecules.

Allosteric inhibition and kinetic characterization of Klebsiella pneumoniae CysE: An emerging drug target

The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity.

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action.

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.

Transcription of genes associated with nickel resistance induced by different doses of nickel nitrate in Quercus rubra

ABSTRACTKnowledge of the mechanism of genetic resistance to nickel (Ni) toxicity in red oak (Quercus rubra) is limited. The main objective of the present study was to evaluate the level of transcription of genes associated with nickel resistance in Q. rubra plants exposed to different doses of potassium nitrate and nickel nitrate. All the Q. rubra genotypes screened were highly resistant to nickel nitrate and potassium nitrate. An unexpected high level of transcription of (ACC) deaminase was induced in leaves by the low dose of potassium nitrate (150 mg/kg). This gene response decreased as the dose was increased to reach the lowest level at the high dose (1600 mg/kg). On the other hand, nickel induced significantly higher level of ACC deaminase transcription only for 1600 mg/kg of nickel nitrate. This transcription was higher in leaves compared to roots. For serine acetyltransferase (SAT) gene, the transcription was higher in roots than in leaves. Surprisingly, potassium nitrate (a common plant fertiliser) induced an upregulation of nicotianamine synthase (NAS3) gene in leaves of samples exposed to 150 mg/kg dose and a downregulation for the 1600 mg/kg treatment. An opposite trend attributed to nickel was observed with nickel nitrate treatments.

MdGGT1 Impacts Apple miR156 Precursor Levels via Ontogenetic Changes in Subcellular Glutathione Homeostasis.

The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of miR156 are not well understood. The glutathione/glutathione disulfide (GSH/GSSG) ratios and GSH levels decline significantly during phase change, which is consistent with miR156 expression in apple (Malus domestica Borkh.). Here, we found that the content of protein conjugated glutathione was remarkably higher in chloroplasts and nuclei of adult than juvenile phase apple hybrids. The decrease in miR156 expression was most relevant to the activities of serine acetyltransferase (SAT) and soluble γ-glutamyl transpeptidase (GGT), and the expressions of MdGGT1 or MdSATs. Transgenic apples over-expressing MdMIR156 or miR156-mimetic (MIM156) did not alter MdGGT1 expression or the soluble GGT activity. Inhibition of GGT activity with serine-borate complex or acivicin led to significant reduction in GSH content, the GSH/GSSG ratio, and the expressions of MdMIR156a5, MdMIR156a12, and miR156. Depletion of GSH with diethyl maleate without altering GGT activity caused a dramatic decrease in the expression of MdMIR156a5, MdMIR156a12, and miR156. Manipulating GGT activity and GSH homeostasis by transgenic over-expressing or RNAi MdGGT1 increased or decreased MdMIR156a5 and MdMIR156a12 levels, respectively. These data provided novel evidence that MdGGT1 participates in transcriptional level of transcription regulation of miR156 precursors during ontogenesis.HIGHLIGHTS- MdGGT1 affects thiol redox status and indirectly participates in the regulation of miR156 expression during vegetative phase change.

The effect of desulfurization on the postharvest quality and sulfite metabolism in pulp of sulfitated "Feizixiao" Litchi (Litchi chinensis Sonn.) fruits.

The residual sulfite caused by sulfur fumigation (SF) is a hazard to health and influenced the export trade of litchi. Desulfurization (DS) is a valid chemical method to reduce the residual sulfite. However, the effect of DS on fumigated litchi has not been studied at physiological and molecular level. This study was aimed to evaluate the effect of DS (SF plus 3% desulfurizer) on the postharvest quality, sulfite residue, and the sulfite metabolism in sulfitated “Feizixiao” litchi during the 4°C storage. Results indicated that the DS promoted the color recovery of sulfitated litchi and achieved an effect similar to SF on controlling rot and browning. DS recovered the water content and respiration rate of sulfitated litchi pericarp. Thus, DS improves commodity properties of sulfitated litchi. Moreover, DS greatly reduced sulfite residue especially in pulp and ensured the edible safety of sulfitated litchi. The activities of sulfite oxidase, sulfite reductase, serine acetyltransferase, and O‐acetylserine(thiol) lyase in pulp increased after SF but fell down after DS while the expressions of their encoding genes decreased after SF but then rallied after DS. These results indicated the key role of these enzymes in sulfite metabolism after SF and DS changed the sulfite metabolism at both enzymatic and transcriptional level. It could be concluded that DS used in this study was an effective method for improving the color recovery and ensuring the edible safety of sulfitated litchi by not only chemical reaction but also both of enzymatic and transcriptional regulation.

Characterization of serine acetyltransferase (CysE) from methicillin-resistant Staphylococcus aureus and inhibitory effect of two natural products on CysE

Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital-acquired infective pathogen that has developed resistance to many antibiotics. It is imperious to develop novel anti-MRSA drugs to control the emergence of drug resistance. The biosynthesis of cysteine in bacteria is catalyzed by CysE and CysK. CysE was predicted to be important for bacterial viability, it could be a potential drug target. The serine acetyltransferase activity of CysE was detected and its catalytic properties were also determined. CysE homology model was built to investigate interaction sites between CysE and substrate L-Ser or inhibitors by molecular docking. Docking data showed that residues Asp94 and His95 were essential for serine acetyltransferase activity of CysE, which were confirmed by site-directed mutagenesis. Colorimetric assay was used to screen natural products and six compounds which inhibited CysE activity (IC50 ranging from 29.83 μM to 203.13 μM) were found. Inhibition types of two compounds 4 (11-oxo-ebracteolatanolide B) and 30 ((4R,4aR)-dihydroxy-3-hydroxymethyl-7,7,10a-trimethyl-2,4,4a,5,6,6a,7,8,9,10,10a,l0b-dodecahydrophenanthro[3,2-b]furan-2-one) on CysE were determined. Compounds 4 and 30 showed inhibitory effect on MRSA growth (MIC at 12.5 μg/ml and 25 μg/ml) and mature biofilm. The established colorimetric assay will facilitate further high-throughput screening of CysE inhibitors from different compound libraries. The compounds 4 and 30 may offer structural basis for developing new anti-MRSA drugs.

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana.

Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants.

Thiol redox-regulation for efficient adjustment of sulfur metabolism in acclimation to abiotic stress.

Sulfur assimilation and sulfur metabolism are tightly controlled at the transcriptional, post-transcriptional, and post-translational levels in order to meet the demand for reduced sulfur in growth and metabolism. These regulatory mechanisms coordinate the cellular sulfhydryl supply with carbon and nitrogen assimilation in particular. Redox homeostasis is an important cellular parameter intimately connected to sulfur by means of multiple thiol modifications. Post-translational thiol modifications such as disulfide formation, sulfenylation, S-nitrosylation, persulfidation, and S-glutathionylation allow for versatile switching and adjustment of protein functions. This review focuses on redox-regulation of enzymes involved in the sulfur assimilation pathway, namely adenosine 5´-phosphosulfate reductase (APR), adenosine 5´-phosphosulfate kinase (APSK), and γ-glutamylcysteine ligase (GCL). The activity of these enzymes is adjusted at the transcriptional and post-translational level depending on physiological requirements and the state of the redox and reactive oxygen species network, which are tightly linked to abiotic stress conditions. Hormone-dependent fine-tuning contributes to regulation of sulfur assimilation. Thus, the link between oxylipin signalling and sulfur assimilation has been substantiated by identification of the so-called COPS module in the chloroplast with its components cyclophilin 20-3, O-acetylserine thiol lyase, 2-cysteine peroxiredoxin, and serine acetyl transferase. We now have a detailed understanding of how regulation enables the fine-tuning of sulfur assimilation under both normal and abiotic stress conditions.

Structural biology of plant sulfur metabolism: from sulfate to glutathione.

Sulfur is an essential element for all organisms. Plants must assimilate this nutrient from the environment and convert it into metabolically useful forms for the biosynthesis of a wide range of compounds, including cysteine and glutathione. This review summarizes structural biology studies on the enzymes involved in plant sulfur assimilation [ATP sulfurylase, adenosine-5'-phosphate (APS) reductase, and sulfite reductase], cysteine biosynthesis (serine acetyltransferase and O-acetylserine sulfhydrylase), and glutathione biosynthesis (glutamate-cysteine ligase and glutathione synthetase) pathways. Overall, X-ray crystal structures of enzymes in these core pathways provide molecular-level information on the chemical events that allow plants to incorporate sulfur into essential metabolites and revealed new biochemical regulatory mechanisms, such as structural rearrangements, protein-protein interactions, and thiol-based redox switches, for controlling different steps in these pathways.

Recombinant production of active Streptococcus pneumoniae CysE in E. coli facilitated by codon optimized BL21(DE3)-RIL and detergent

The emergence of drug resistance in Streptococcus pneumoniae (Spn) is a global health threat and necessitates discovery of novel therapeutics. The serine acetyltransferase (also known as CysE) is an enzyme of cysteine biosynthesis pathway and is reported to be essential for the survival of several pathogenic bacteria. Therefore, it appears to be a very attractive target for structure–function understanding and inhibitor design. This study describes the molecular cloning of cysE from Spn in the pET21c vector and efforts carried out for expression and purification of active recombinant CysE. Significant expression of recombinant Spn cysE could be achieved in codon optimized BL21(DE3)-RIL strain as opposed to conventional BL21(DE3) strain. Analysis of codon adaptation index (CAI) with levels of eukaryotic genes and prokaryotic cysEs expressed in heterologous E. coli host suggests that codon optimized E. coli BL21(DE3)-RIL may be a better host for expressing genes with low CAI. Here, an efficient protocol has been developed for recovery of recombinant Spn CysE in soluble and biologically active form by the usage of nonionic detergent Triton X-100 at a concentration as low as 1%. Altogether, this study reports a simple strategy for producing functionally active Spn CysE in E. coli.

Selenium enhances antioxidant capacity by improving dencichine content in Panax notoginseng

Dencichine is the main ingredient in Panax notoginseng, which is an herb that plays a key role in hemostasis. The biosynthesis of dencichine requires the participation of sulfur. Selenium and sulfur are congeners. The purpose of this study was to reveal if selenium participates in the synthesis of dencichine, and to determine whether dencichine plays a role in anti-stress effects. P. notoginseng and planting soil were sampled from different sites and subjected to selenium and dencichine determinations. Biosynthesis of dencichine was investigated by exposing the seedlings to different selenium concentrations in pot and hydroponic experiments. The effects of dencichine on the regulation of the antioxidant enzyme system and reactive oxygen species (ROS) were also tested in hydroponic experiments. The results indicated that the selenium and dencichine contents of roots were 0.69-0.98% and 0.21–1.18 mg kg−1, respectively, and the soil selenium content in 18 planting areas in Yunnan Province was 0.16-0.32 mg kg−1. Dencichine and selenium contents in soil or plants were positively correlated (R2 = 0.1643***, R2 = 0.3084***, respectively). Selenium treatment increased the dencichine content by 69–111% in pot experiments using 5 mg kg−1 and by 23–33% in hydroponic experiments using 2 μM. The key genes in the dencichine biosynthetic pathway, such as β-cyanoalanine synthase and serine acetyltransferase, were significantly upregulated by the 2 μM selenium treatment. Dencichine treatment (30 μM) significantly enhanced root activity, increased antioxidant capacity, decreased ROS content, and reduced lipid peroxidation of the cell membranes. Therefore, selenium promoted the synthesis of dencichine, which acted as an anti-stress agent in P. notoginseng. However, the selenium content was deficient in the P. notoginseng planting soil from Yunnan Province. These results suggest that appropriate selenium supplementation is beneficial for the growth and development of P. notoginseng and the accumulation of dencichine.

Insights into multifaceted activities of CysK for therapeutic interventions.

CysK (O-acetylserine sulfhydrylase) is a pyridoxal-5' phosphate-dependent enzyme which catalyzes the second step of the de novo cysteine biosynthesis pathway by converting O-acetyl serine (OAS) into l-cysteine in the presence of sulfide. The first step of the cysteine biosynthesis involves formation of OAS from serine and acetyl CoA by CysE (serine acetyltransferase). Apart from role of CysK in cysteine biosynthesis, recent studies have revealed various additional roles of this enzyme in bacterial physiology. Other than the suggested regulatory role in cysteine production, other activities of CysK include involvement of CysK-in contact-dependent toxin activation in Gram-negative pathogens, as a transcriptional regulator of CymR by stabilizing the CymR-DNA interactions, in biofilm formation by providing cysteine and via another mechanism not yet understood, in ofloxacin and tellurite resistance as well as in cysteine desulfurization. Some of these activities involve binding of CysK to another cellular partner, where the complex is regulated by the availability of OAS and/or sulfide (H2S). The aim of this study is to present an overview of current knowledge of multiple functions performed by CysK and identifying structural features involved in alternate functions. Due to possible role in disease, promoting or inhibiting a "moonlighting" function of CysK could be a target for developing novel therapeutic interventions.

Metabolic engineering of Corynebacterium glutamicum for L-cysteine production.

L-cysteine, a valuable sulfur-containing amino acid, has been widely used in food, agriculture, and pharmaceutical industries. Due to the toxicity and complex regulation of L-cysteine, no efficient cell factory has yet been achieved for L-cysteine industrial production. In this study, the food-grade microorganism Corynebacterium glutamicum was engineered for L-cysteine production. Through deletion of the L-cysteine desulfhydrases (CD) and overexpression of the native serine acetyltransferase (CysE), the initial L-cysteine-producing strain CYS-2 was constructed to produce 58.2 ± 5.1mg/L of L-cysteine. Subsequently, several metabolic engineering strategies were performed to further promote L-cysteine biosynthesis, including using strong promoter tac-M to enhance expression intensity of CysE, investigating the best candidate among several heterogeneous feedback-insensitive CysEs for L-cysteine biosynthesis, overexpressing L-cysteine synthase (CysK) to drive more metabolic flux, evaluating the efflux capacity of several heterogeneous L-cysteine transporters, engineering L-serine biosynthesis module to increase the precursor L-serine level and using thiosulfate as the sulfur source. Finally, the L-cysteine concentration of the engineered strain CYS-19 could produce 947.9 ± 46.5mg/L with addition of 6g/L Na2S2O3, approximately 14.1-fold higher than that of the initial strain CYS-2, which was the highest titer of L-cysteine ever reported in C. glutamicum. These results indicated that C. glutamicum was a promising platform for L-cysteine production.

Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites.

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a cysteine-dependent manner with relatively low mammalian cytotoxicity.