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Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

Sulfur compounds are known to play crucial roles in plants facilitating tolerance to heavy metal stress. Previous studies have shown the protective role of exogenous cysteine (Cys) to improve chromium (Cr) tolerance. In the present study, we aimed to evaluate the capability of Cys (0.5 mM) to reduce chromium toxicity (100 μM) in rapeseed by physiological and proteomic analyses. Seedlings treated with Cr showed reduced growth and activities of antioxidant enzymes, and increased malondialdehyde (MDA) content. Exogenous application of Cys reduced Cr accumulation and MDA content, restored catalase (CAT) and peroxidase (POD) activities, and caused significant enhancements in seedling growth. 2-DE gel electrophoresis and mass spectrometry were performed to investigate changes in the root proteome in response to Cys and Cr. The Cr-induced down-regulations of glycine-rich RNA-binding protein, lactoylglutathione lyase, fructose-bisphosphate aldolase, and glutamine synthetase were restored with Cys. Additionally, Cys removed the Cr-induced up-regulation of some proteins including pathogenesis-related protein, major latex protein (MLP) like proteins, 22.0 kDa HSP, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, glutamine amidotransferase, cysteine synthase, and L-lactate dehydrogenase. Taken together, proteomic analysis revealed that exogenous Cys helps plants to overcome Cr stress by modulating energy metabolism, amino acid metabolism, and defense mechanism.

Plants catalyze the biosynthesis of a large number of non-protein amino acids, which are usually toxic for other organisms. In this review, the chemistry and metabolism of N-heterocyclic non-protein amino acids from plants are described. These N-heterocyclic non-protein amino acids are composed of β-substituted alanines and include mimosine, β-pyrazol-1-yl-L-alanine, willardiine, isowillardiine, and lathyrine. These β-substituted alanines consisted of an N-heterocyclic moiety and an alanyl side chain. This review explains how these individual moieties are derived from their precursors and how they are used as the substrate for biosynthesizing the respective N-heterocyclic non-protein amino acids. In addition, known catabolism and possible role of these non-protein amino acids in the actual host is explained.

Pyridoxal-5'-phosphate-dependent cysteine synthases synthesize l-cysteine from their primary substrates, O-acetyl-l-serine (OAS) and O-phospho-l-serine (OPS), and their secondary substrate, sulfide. The mechanism by which cysteine synthases recognize OPS remains unclear; hence, we investigated the OPS recognition mechanism of the OPS sulfhydrylase obtained from Aeropyrum pernix K1 (ApOPSS) and the OAS sulfhydrylase-B obtained from Escherichia coli (EcOASS-B), using protein engineering methods. From the amino acid sequence alignment data, we found that some OPS sulfhydrylases (OPSSs) had a Tyr corresponding to the Phe225 and Phe141 residues in ApOPSS and EcOASS-B, respectively, and that the Tyr residue could facilitate OPS recognition. The enzymatic activity of the ApOPSS F225Y mutant toward OPS decreased compared with that of the wild-type; the kcat value decreased 2.3-fold during cysteine synthesis. X-ray crystallography results of the complex of ApOPSS F225Y and F225Y/R297A mutants bound to OPS and l-cysteine showed that kcat might have decreased because of the stronger interactions of the reaction product phosphate with Tyr225, Thr203, and Arg297, and that of the l-cysteine with Tyr225. The specific activity of the EcOASS-B F141Y mutant toward OPS increased by 50-fold compared with that of the wild-type. Thus, a Tyr within a cysteine synthase corresponding to the Phe225 in ApOPSS and Phe141 in EcOASS-B could act as a key residue for classifying an unknown cysteine synthase as an OPSS. The elucidation of the substrate recognition system of cysteine synthases would enable us to effectively classify cysteine synthases and develop pathogen-specific drug targets, as OPSS is absent in mammalian hosts.

Antibiotics are the cornerstone of modern medicine and agriculture, and rising antibiotic resistance is one the biggest threats to global health and food security. Identifying new and different druggable targets for the development of new antibiotics is absolutely crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non-essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is crucial for the synthesis of many biomolecules important for defense against the host immune system. Cysteine synthesis is a two-step process, catalyzed by two enzymes. Serine acetyltransferase (CysE) catalyzes the first step to synthesize the pathway intermediate O-acetylserine, and O-acetylserine sulfhydrylase (CysK/CysM) catalyzes the second step using sulfide or thiosulfate to produce cysteine. Disruption of the cysteine biosynthesis pathway results in dysregulated sulfur metabolism, altering the redox state of the cell leading to decreased fitness, enhanced susceptibility to oxidative stress and increased sensitivity to antibiotics. In this review, we summarize the structure and mechanism of characterized CysE and CysK/CysM enzymes from a variety of bacterial pathogens, and the evidence that support targeting these enzymes for the development of new antimicrobials or antibiotic adjuvants. In addition, we explore and compare compounds identified thus far that target these enzymes.

Pyridoxal-5'-phosphate (PLP)-dependent enzymes have garnered interest for their ability to synthesize non-standard amino acids (nsAAs). One such class of enzymes, O-acetylserine sulfhydrylases (OASSs), catalyzes the final step in the biosynthesis of l-cysteine. Here, we examine the β-substitution capability of the OASS from Citrullus vulgaris (CvOASS), a putative l-mimosine synthase. While the previously reported mimosine synthase activity was not reproducible in our hands, we successfully identified non-native reactivity with a variety of O-nucleophiles. Optimization of reaction conditions for carboxylate and phenolate substrates led to distinct conditions that were leveraged for the preparative-scale synthesis of nsAAs. We further show this enzyme is capable of C-C bond formation through a β-alkylation reaction with an activated nitroalkane. To facilitate understanding of this enzyme, we determined the crystal structure of the enzyme bound to PLP as the internal aldimine at 1.55 Å, revealing key features of the active site and providing information that may guide subsequent development of CvOASS as a practical biocatalyst.

Horizontal gene transfer (HGT) plays an important role in evolutionary processes as organisms adapt to their environments, and now cases of gene duplication after HGT in eukaryotes are emerging at an increasing rate. However, the fate and roles of the duplicated genes over time in eukaryotes remain unclear. Here we conducted a comprehensive analysis of the evolution of cysteine synthase (CYS) in lepidopteran insects. Our results indicate that HGT-derived CYS genes are widespread and have undergone duplication following horizontal transfer in many lepidopteran insects. Moreover, lepidopteran CYS proteins not only have β-cyanoalanine synthase activity but also possess cysteine synthase activity that is involved in sulfur amino acid biosynthesis. Duplicated CYS genes show marked divergence in gene expression patterns and enzymatic properties, suggesting that they probably have undergone subfunctionalization and/or neofunctionalization in Lepidoptera. The gene transfer of CYS genes and subsequent duplication appears to have facilitated the adaptation of lepidopteran insects to different diets and promoted their ecological diversification. Overall, this study provides valuable insights into the ecological and evolutionary contributions of CYS in lepidopteran insects.

β-N-Oxalyl-l-α,β-diaminopropionic acid (β-ODAP), found in Lathyrus sativus at first, causes a neurological disease, lathyrism, when over ingested in an unbalanced diet. Our previous research suggested that β-ODAP biosynthesis is related to sulfur metabolism. In this study, β-cyanoalanine synthase (β-CAS) was confirmed to be responsible for β-ODAP biosynthesis via in vitro enzymatic analysis. LsCAS was found to be pyridoxal phosphate (PLP)-dependent via spectroscopic analysis and dual functional via enzymatic activity analysis. Generation of a M135T/M235S/S239T triple mutant of LsCAS, which are the key sites to control the ratio of CAS/cysteine synthase (CS) activity, switches reaction chemistry to that of a CS. LsCAS interactions were further screened and verified via Y2H, BiFC and pull-down assay. It was suggested that LsSAT2 interacts and forms a cysteine regulatory complex (CRC) with LsCAS in mitochondria, which improves LsSAT while reduces LsCAS activities to affect β-ODAP content positively. These results provide new insights into the molecular regulation of β-ODAP content in L. sativus.

Serine synthase (SS) from Fusobacterium nucleatum is a fold type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine with water to form l-serine and H2S. Herein, we show that SS can also function as a cysteine synthase, catalyzing the β-replacement of l-serine with bisulfide to produce l-cysteine and H2O. The forward (serine synthase) and reverse (cysteine synthase) reactions occur with comparable turnover numbers and catalytic efficiencies for the amino acid substrate. Reaction of SS with l-cysteine leads to transient formation of a quinonoid species, suggesting that deprotonation of the Cα and β-elimination of the thiolate group from l-cysteine occur via a stepwise mechanism. In contrast, the quinonoid species was not detected in the formation of the α-aminoacrylate intermediate following reaction of SS with l-serine. A key active site residue, D232, was shown to stabilize the more chemically reactive ketoenamine PLP tautomer and also function as an acid/base catalyst in the forward and reverse reactions. Fluorescence resonance energy transfer between PLP and W99, the enzyme's only tryptophan residue, supports ligand-induced closure of the active site, which shields the PLP cofactor from the solvent and increases the basicity of D232. These results provide new insight into amino acid metabolism in F. nucleatum and highlight the multiple catalytic roles of D232 in a new member of the fold type II family of PLP-dependent enzymes.

Aminooxyacetate (AOA) is a pyridoxal phosphate antagonist that inhibits various plant enzymes (including transaminases) which require pyridoxal phosphate as a cofactor and it exhibits phytotoxic and herbicidal properties. We examined AOA and its analog, N-t-butoxycarbonyl-AOA (Boc-AOA) for phytotoxicity, interactions with weed pathogens (bioherbicides), and effects on an important pyridoxal requiring enzyme, cysteine synthase (CS, E.C. 4.2.99.8). Studies were performed on two weeds, i.e., hemp sesbania [Sesbania exaltata (Raf.) Rybd. Ex A.W. Hill] and sicklepod (Senna obtusifolia), and two pathogens, (Colletotrichum truncatum and Alternaria cassiae), that are bioherbicidal agents against hemp sesbania and sicklepod, respectively. Pathogenicity tests, and assays for extractable, and in vitro CS activities were utilized. Phytotoxicity bioassays indicated that the bulky t-butoxycarbonyl moiety substitution on the AOA molecule did not substantially hinder expression of biological activity of Boc-AOA in these tests. Generally, spray application of the compounds to young dark-grown seedlings caused little growth effects, but root-feeding of the chemicals reduced growth (stem elongation) in both weeds. Hemp sesbania was generally more tolerant than sicklepod to these compounds. The only apparent positive interaction of the chemicals with these pathogens was the Boc-AOA: C. truncatum combination treatment on hemp sesbania. Both compounds reduced extractable CS in the seedlings by 30%, 72 h after treatment. CS activity was reduced by 15% in hemp sesbania treated with C. truncatum but increased 20% above control levels after infection of sicklepod by A. cassiae. This latter effect suggests that CS may be involved in sicklepod defense mechanisms against this pathogen.

Edwardsiella piscicida (E. piscicida) is an important fish pathogen. However, the mechanism of Glu6P transport regulatory protein UhpA how to affect the virulence gene expression in E. piscicida is still unclear. The results in this study showed that the metabolism-related gene expression of cysteine synthase (orf 1134) and sulphate transporter (ychM) in the uhpA mutant strain ΔuhpA was 0.76-fold and 0.68-fold lower than the ones in the wild strains (p < .05). The gene expression of ethA and ethB in the ΔuhpA strain was 0.80-fold and 0.72-fold lower than the ones in the wild strains (p < .05). However, the gene expression of fliC and flgN in the ΔuhpA was 1.51-fold and 1.21-fold higher than the ones in the wild strains (p < .05). The gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the ΔuhpA was 1.27-fold, 1.13-fold, 1.28-fold and 1.23-fold higher than the ones in the wild strains (p < .05). This suggested that the uhpA gene could regulate the key virulence gene expression, and the uhpA gene was associated with the pathogenicity of E. piscicida in fish.

Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grains. Arsenic detoxification is closely linked to sulfur assimilation, but the genes involved have not been described in rice. In this study, we characterize the function of OASTL-A1, an O-acetylserine(thiol) lyase, in cysteine biosynthesis and detoxification of As in rice. Tissue expression analysis revealed that OsOASTL-A1 is mainly expressed in roots at the vegetative growth stage and in nodes at the reproductive stage. Furthermore, the expression of OsOASTL-A1 in roots was strongly induced by As exposure. Transgenic rice plants expressing pOsOASTL-A1::GUS (β-glucuronidase) indicated that OsOASTL-A1 was strongly expressed in the outer cortex and the vascular cylinder in the root mature zone. Subcellular localization using OsOASTL-A1:eGFP (enhanced green fluorescent protein) fusion protein showed that OsOASTL-A1 was localized to the cytosol. In vivo and in vitro enzyme activity assays showed that OsOASTL-A1 possessed the O-acetylserine(thiol) lyase activity. Knockout of OsOASTL-A1 led to significantly lower levels of cysteine, glutathione, and phytochelatins in roots and increased sensitivity to arsenate stress. Furthermore, the osoastl-a1 knockout mutants reduced As accumulation in the roots, but increased As accumulation in shoots. We conclude that OsOASTL-A1 is the cytosolic O-acetylserine(thiol) lyase that plays an important role in non-protein thiol biosynthesis in roots for As detoxification.

The purslane Portulaca oleraceae L. can tolerant high saline stress and has been developed as a seawater-irrigated vegetable. Currently, the underlying molecular mechanisms were still not well understood. In the present study, purslane seedlings were treated with 200 mM NaCl and then stems were subjected to transcriptome sequencing and metabolome analyses. Together with real-time quantitative PCR validation, the results showed that treatment with 200 mM NaCl depressed expression levels of genes for photosynthesis and aquaporins, suggesting that saline stress might inhibit photosynthesis and water uptake. However, expression levels of aminocyclopropane-1-carboxylic acid synthase, bifunctional L-3-cyanoalanine synthase/cysteine synthase and cyanoalanine synthase increased, suggesting elevation of ethylene production, which were further transduced by ethylene-responsive receptor and ethylene-insensitive 3 transcription factor. Metabolome analyses detected higher contents of pyrophosphate, D-galacturonic acid and elaidic acid in treatment with 200 mM NaCl than those in the control. These changes should positively regulate glycolysis, energy supply and integrity of cell membrane. Overall, changes of ethylene, pyrophosphate and elaidic acid might facilitate adaptation of purslane to saline stress.

Tetranychus urticae is a polyphagous spider mite that can feed on more than 1100 plant species including cyanogenic plants. The herbivore genome contains a horizontally acquired gene tetur10g01570 (TuCAS) that was previously shown to participate in cyanide detoxification. To understand the structure and determine the function of TuCAS in T. urticae, crystal structures of the protein with lysine conjugated pyridoxal phosphate (PLP) were determined. These structures reveal extensive TuCAS homology with the beta-substituted alanine synthase family, and they show that this enzyme utilizes a similar chemical mechanism involving a stable alpha-aminoacrylate intermediate in beta-cyanoalanine and cysteine synthesis. We demonstrate that TuCAS is more efficient in the synthesis of beta-cyanoalanine, which is a product of the detoxification reaction between cysteine and cyanide, than in the biosynthesis of cysteine. Also, the enzyme carries additional enzymatic activities that were not previously described. We show that TuCAS can detoxify cyanide using O-acetyl-L-serine as a substrate, leading to the direct formation of beta-cyanoalanine. Moreover, it catalyzes the reaction between the TuCAS-bound alpha-aminoacrylate intermediate and aromatic compounds with a thiol group. In addition, we have tested several compounds as TuCAS inhibitors. Overall, this study identifies additional functions for TuCAS and provides new molecular insight into the xenobiotic metabolism of T. urticae.

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 41.54 kDa. 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 ± 26 µM and 72.83 s−1 for OAS, respectively, and 60 ± 2 µM and 2.43 s−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.

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.

Cysteine is a crucial component for all organisms and plays a critical role in the structure, stability, and catalytic functions of many proteins. Tetrahymena has reverse transsulfuration and de novo pathways for cysteine biosynthesis. Cysteine synthase is involved in the de novo cysteine biosynthesis and catalyzes the production of cysteine from O-acetylserine. The novel cysteine synthase TtCSA2 was identified from Tetrahymena thermophila. The TtCSA2 showed high expression levels at the log-phase and the sexual development stage. The TtCsa2 was localized on the outer mitochondrial membrane throughout different developmental stages. However, the truncated N-terminal signal peptide mutant TtCsa2-ΔN23 was localized into the mitochondria. His-TtCsa2 was expressed in Escherichia coli and purified using affinity chromatography. The His-TtCsa2 showed O-acetylserine sulfhydrylase and serine sulfhydrylase activities. Cysteine and glutathione contents decreased in the csa2KD mutant. Furthermore, mutant cells were sensitive to cadmium and copper stresses. This study indicated that the TtCSA2 was involved in the cysteine synthesis in mitochondria and related to heavy metal stresses resistance in Tetrahymena.

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.

Recent years have witnessed tremendous progress in understanding the biological relevance of hydrogen sulfide (H2S) as the third gasotransmitter for multiple signaling events in plants. l-Cys desulfhydrase (LCD) catalyzes the formation of H2S by using l-Cys as a substrate. In previous studies, we detected LCD activity in rice, but the corresponding genetic information and its biological function in the alleviation of plant Cd toxicity are still unknown. In this study, a putative LCD gene (OsLCD2) with high homology to Arabidopsis LCD has been cloned in rice. Surprisingly, the biochemical characterization of the recombinant OsLCD2 protein suggests that this protein predominantly possesses cysteine synthesis activity rather than the degradation of cysteine to produce H2S, and thus, the gene has been renamed OsCS1. OsCS1 localizes inside the nucleus in rice protoplasts. Molecular evidence shows that OsCS1 mRNA is ubiquitously expressed in rice, and up-regulated by cadmium. It has been further observed that over-expression of OsCS1 in Arabidopsis results in the enhanced glutathione biosynthesis, which allows plants to tolerate cadmium stress. Overall, the above findings provide evidence that OsCS1 is a potentially important enzyme responsible for the synthesis of cysteine, which could provide a new information about the metabolism of H2S in plants.

Fusobacterium nucleatum is a common oral bacterium and a major producer of H2S, a toxic gas linked to the pathogenesis of periodontal disease. The bacterium encodes a fold type II pyridoxal l-phosphate (PLP)-dependent enzyme, Fn1220 or lanthionine synthase (LS), that generates H2S and l-lanthionine (a component of the peptidoglycan layer) through β-replacement of l-cysteine by a second molecule of l-cysteine. Herein, we show through detailed kinetic analysis that LS elicits catalytic promiscuity as demonstrated for other fold type II PLP-dependent homologues, namely, O-acetylserine sulfhydrylase (OASS) and cystathionine β-synthase (CBS). Like OASS, LS can assimilate H2S by catalyzing the β-replacement of O-acetyl-l-serine by sulfide to form l-cysteine. However, the turnover for this reaction in LS is slower than that of other studied OASS enzymes due to slower conversion to the α-aminoacrylate intermediate. Similar to yeast and human CBS, LS can generate H2S and l-cystathionine through β-replacement of l-cysteine by a second molecule of l-homocysteine; however, whereas this is the main H2S-forming reaction in CBS, it is not for LS. LS shows a marked preference for forming H2S and l-lanthionine through the condensation of 2 equiv of l-cysteine. Sequence alignment of LS with other CBS and OASS enzymes and inspection of the LS crystal structure in the external aldimine state with l-lanthionine reveal that LS possesses a unique loop that engages in hydrogen-bond contact with the product, providing a structural rationale for the enzyme's catalytic preference for H2S and l-lanthionine biosynthesis.

The suboptimal content of sulfur-containing amino acids methionine and cysteine prevents common bean (Phaseolus vulgaris) from being an excellent source of protein. Nutritional improvements to this significant crop require a better understanding of the biosynthesis of sulfur-containing compounds including the nonproteogenic amino acid S-methylcysteine and the dipeptide γ-glutamyl-S-methylcysteine, which accumulate in seed. In this study, seeds were incubated with isotopically labelled serine, cysteine or methionine and analyzed by reverse phase chromatography-high resolution mass spectrometry to track stable isotopes as they progressed through the sulfur metabolome. We determined that serine and methionine are the sole precursors of free S-methylcysteine in developing seeds, indicating that this compound is likely to be synthesized through the condensation of O-acetylserine and methanethiol. BSAS4;1, a cytosolic β-substituted alanine synthase preferentially expressed in developing seeds, catalyzed the formation of S-methylcysteine in vitro. A higher flux of labelled serine or cysteine was observed in a sequential pathway involving γ-glutamyl-cysteine, homoglutathione and S-methylhomoglutathione, a likely precursor to γ-glutamyl-S-methylcysteine. Preferential incorporation of serine over cysteine supports a subcellular compartmentation of this pathway, likely to be in the chloroplast. The origin of the methyl group in S-methylhomoglutathione was traced to methionine. There was substantial incorporation of carbons from methionine into the β-alanine portion of homoglutathione and S-methylhomoglutathione, suggesting the breakdown of methionine by methionine γ-lyase and conversion of α-ketobutyrate to β-alanine via propanoate metabolism. These findings delineate the biosynthetic pathways of the sulfur metabolome of common bean and provide an insight that will aid future efforts to improve nutritional quality.

Soil salinization is one major constraint to plant geographical distribution, yield, and quality, and as an ideal plant for the "greening" of flat-roofed buildings, Sedum lineare Thunb. has strong tolerance against a variety of environmental adversities including salinity with the underlying mechanism still remaining unknown. In this study, we performed de novo transcriptome sequencing on leaf and root samples of NaCl-treated S. lineare Thunb. and identified 584 differentially expressed genes (DEGs), which were further annotated by gene function classification and pathway assignments using the public data repositories. In addition to the increased gene expression level verified by qRT-PCR, the elevated activities of the corresponding enzymes were also demonstrated for peroxidase (POD), glutathione peroxidases (GPX), and cysteine synthase (CSase) in the NaCl-treated roots. Furthermore, two highly inducible genes without known functions related to salt tolerance were selected to be overexpressed and tested for their effects on salt tolerance in the model plant, Arabidopsis thaliana. Upon 150 mM NaCl treatment, 35S:SlCXE but not 35S:SlCYP72A transgenic Arabidopsis seedlings exhibited improved salt resistance as shown by the increased seed germination rates and longer primary roots of transgenic seedlings when compared to wild-type plants. Taken together, this work laid a foundation for a better understanding of the salt adaptation mechanism of S. lineare Thunb. and genes identified could serve as useful resources for the development of more salt-tolerant varieties of other species through genetic engineering.

Hydrogen sulfide (H2S) is one of the key gasotransmitters in plant and animal cells. The term “hydrogen sulfide” means not only H2S as a dissolved gaseous compound, but also the HS– anion, into which, under physiologically normal conditions, about 80% of molecular hydrogen sulfide is converted. One of the most notable hydrogen sulfide physiological effects is the activation of adaptive plant responses. However, the activation mechanisms of plant stress-protective systems under the H2S influence, direct targets of its action, signaling and hormonal mediators providing physiological effects, remain poorly understood. Analysis and systematization of information on hydrogen sulfide synthesis, signaling, and activation of adaptive reactions with its participation became the aim of this review. Hydrogen sulfide synthesis. To date, it has been found that in plants hydrogen sulfide can be synthesized using six enzymes (See Fig. 1). Conversion of L-cysteine to pyruvate catalyzed by L-cysteine desulfhydrase with release of hydrogen sulfide and ammonium is considered as one of the main ways of synthesizing H2S in plants. It is also possible to form hydrogen sulfide from D-cysteine under the action of D-cysteine desulfhydrase. Hydrogen sulfide can also be synthesized by sulfite reduction with the participation of sulfite reductase. Formation of hydrogen sulfide in plants involving β-cyanoalanine synthase, cysteine synthase and carbonic anhydrase is also expected. Hydrogen sulfide signaling. Hydrogen sulfide does not have specific molecular receptors. It is assumed that primary molecular effects of H2S are associated with S-sulfhydration (persulfidation) – conversion of –SH cysteine residues to –SSH. The most common proteins whose state is regulated by sulfhydration are peroxyredoxins, which, in turn, are among the key participants in cellular redox regulation. Hydrogen sulfide is also involved in processes of redox regulation occuring with participation of reactive oxygen species (ROS) and nitric oxide (NO), and can affect cell calcium homeostasis (See Fig. 2). In this case, however, the sequence of arrangement of these intermediaries in formation of various adaptive reactions of plants in many cases remains unknown. There is evidence of an increase in the ROS content in plant cells under the influence of hydrogen sulfide, due, primarily, to the activation of NADPH oxidase. At the same time, synthesis of hydrogen sulfide can be induced by an action of hydrogen peroxide on plant objects. Hydrogen sulfide can directly and indirectly influence activity and expression of antioxidant enzyme genes, which also affects cell redox homeostasis. It was shown that hydrogen sulfide and nitric oxide can act on the same protein targets, causing effects of persulfidation or nitrosylation. Moreover, NO and H2S also affect the intracellular content of each other. Hydrogen sulfide is in a rather complex functional interaction with calcium ions. Activation of hydrogen sulfide synthesis associated with increased expression of L-cysteine desulfhydrase gene can be induced with calcium and calmodulin. On the other hand, hydrogen sulfide can cause an opening of calcium channels of plant cells. Hydrogen sulfide interacts with a complex network of hormonal signaling too (See Fig. 2). In particular, its synthesis can be induced by abscisic acid (ABA). On the other hand, H2S can mediate physiological effects of ABA. Hydrogen sulfide can activate synthesis of jasmonic acid in plants. Also, H2S is involved in plant adaptive reactions induction under influence of salicylic acid and polyamines. In general, hydrogen sulfide is involved in a complex regulatory network of signaling and hormonal mediators. Participation in plant adaptation. In response to the impact of many stressors (high and low temperatures, dehydration, salinization), the content of endogenous hydrogen sulfide in plants increases. Moreover, mutants defective in the hydrogen sulfide synthesis were not resistant to action of these stress factors. Plant treatment with hydrogen sulfide donors (in particular, sodium hydrosulfide NaHS) increases resistance of plants to stress temperatures, drought, salt stress, action of heavy metals, UV-B and other factors (See Table). Hydrogen sulfide has a pronounced activating effect on expression of antioxidant enzyme genes, accumulation of polyfunctional low-molecular-weight protective compounds, in particular proline and sugars. Of particular importance for plant adaptation is the accumulation under the influence of hydrogen sulfide of a wide range of secondary metabolites, including phenolic compounds and flavonoids, which have a pronounced antioxidant effect. Hydrogen sulfide is also involved in regulation of plant stomatal reactions. Dependence of the stomata closing process under osmotic and salt stress on the activity of cysteine desulfhydrase and H2S synthesis was shown. The effect of hydrogen sulfide on stomatal aperture, as well as other stomata closing inducers, is associated with a change in the ion channels state, in particular, potassium channels (K+ out) of guard cells. ROS, calcium ions and, possibly, components of lipid signaling are involved in the implementation of these effects of hydrogen sulfide. Hydrogen sulfide donors can be used not only to induce adaptive reactions of plants, but also in storage technology for agricultural products. The use of NaHS during storage of fruit and berries prevents their ripening and aging, contributes to the preservation of a pool of antioxidants, in particular, ascorbic acid, phenolic compounds and flavonoids. Also, hydrogen sulfide can be used to extend the life of cut flowers. A further study of stress-protective effects of hydrogen sulfide will allow, on the one hand, to more deeply understand the adaptation mechanisms, and, on the other hand, to create theoretical foundations for new approaches in agrobiotechnology.

We present the genome sequence of Saccharospirillum mangrovi HK-33T, isolated from a mangrove sediment sample in Haikou, China. The complete genome of S. mangrovi HK-33T consisted of a single-circular chromosome with the size of 3,686,911 bp as well as an average G + C content of 57.37%, and contained 3,383 protein-coding genes, 4 operons of 16S-23S-5S rRNA genes, and 52 tRNA genes. Genomic annotation indicated that the genome of S. mangrovi HK-33T had many genes related to oligosaccharide and polysaccharide degradation and utilization of polyhydroxyalkanoate. For nitrogen cycle, genes encoding nitrate and nitrite reductase, glutamate dehydrogenase, glutamate synthase, and glutamine synthetase could be found. For phosphorus cycle, genes related to polyphosphate kinases (ppk1 and ppk2), the high-affinity phosphate-specific transport (Pst) system, and the low-affinity inorganic phosphate transporter (pitA) were predicted. For sulfur cycle, cysteine synthase and type III acyl coenzyme A transferase (dddD) coding genes were searched out. This study provides evidence about carbon, nitrogen, phosphorus, and sulfur metabolic patterns of S. mangrovi HK-33T and broadens our understandings about ecological roles of this bacterium in the mangrove sediment environment.

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 ± 5.1 mg/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 ± 46.5 mg/L with addition of 6 g/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.

Hydrogen sulfide (H(2)S) is a key metabolite in biosynthesis and is increasingly being recognized as an essential gasotransmitter. Owing to its diffusible and reactive nature, H(2)S can be difficult to quantify, particularly in situ. Although several detection schemes are available, they have drawbacks. In efforts to quantify sulfide release in the cross-linking reaction of the flagellar protein FlgE we developed an enzyme-coupled sulfide detection assay using the Escherichia coli O-acetylserine sulfhydrylase enzyme CysM. Conversion of HS(−) to L-cysteine via CysM followed by derivatization with the thiol-specific fluorescent dye 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin enables for facile detection and quantification of H(2)S by fluorescent HPLC. The assay was validated by comparison to the well-established methylene blue sulfide detection assay and the robustness demonstrated by interference assays in the presence of common thiols such as glutathione, 2-mercaptoethanol, dithiothreitol and L-methionine, as well as a range of anions. We then applied the assay to the aforementioned lysinoalanine cross-linking by the Treponema denticola flagellar hook protein FlgE. Overall, unlike previously reported H(2)S detection methods, the assay provides a biologically compatible platform to accurately and specifically measure hydrogen sulfide in situ, even when it is produced on long time scales.

Bordetella sp. HZ20 was isolated from the surface of brown seaweed (Laminaria japonica) and absence of the abilities to decompose the brown seaweed. The genome of Bordetella sp. HZ20 was sequenced and comprised of one circular chromosome with the size of 4,227,194 bp and DNA G + C content of 55.5%. Genomic annotation showed that, Bordetella sp. HZ20 may have chitin degradation related enzymes, heparin-sulfate lyase-like protein and enzymes related to the synthase and utilization of polyhydroxyalkanoate for carbon utilization, nitrate and nitrite reductase, glutamate dehydrogenase, glutamate synthase and glutamine synthetase for nitrogen cycle, polyphosphate kinases (pkk1 and pkk2), the high-affinity phosphate-specific transport (Pst) system and the low-affinity inorganic phosphate transporter (pitA) for phosphorus cycle, cysteine synthase and type III acyl coenzyme A transferase (dddD) for sulfur cycle. These features indicated the metabolic patterns of Bordetella sp. HZ20 in C, N, P and S cycles. In addition, the predicted Pst system and cysteine synthase were also related to biofilm formation which showed the potential pathogenicity of Bordetella sp. HZ20 to the cells of animals or plants. This study provides evidences about the metabolic patterns of Bordetella sp. HZ20 and broadens our understandings about ecological roles of bacterium without algal-polysaccharides degrading abilities in the brown seaweed-abundant environment.

The paradoxical effect of deletion of the Escherichia coli genes cysK and cysM encoding cysteine synthase enzymes has been studied: such cysteine auxotrophs actively degrade the excess of cysteine transported from the medium to form H2S. We have shown that deletions of any of the known genes controlling the degradation of exogenous cysteine, including the genes aspC, mstA, cysK, cysM, tnaA, metC, and malY, as well as the newly discovered genes yciW, cyuA, cyuP, and cyuR, do not deprive the cysteine auxotrophs ΔcysK ΔcysM of the ability to degrade cysteine. Cysteine degradation in the ΔcysK ΔcysM mutant is positively regulated by the products of the cysB and cysE genes. It is significant that the ΔcysK ΔcysM mutant shows an increased transcription of the genes opposing the oxidative stress (sodA, catG, arcA, and cydD). We assume that oxidative stress in cells of the ΔcysK ΔcysM mutant is provoked by restriction of cysteine resynthesis, while cysB-dependent degradation of exogenous cysteine and generated H2S provide protection against oxidative stress.

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.

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.

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β‐synthase and cystathionine γ‐lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5′‐phosphate‐dependent enzymes, cystathionine β‐synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N‐terminal heme binding domain, catalytic domain, and C‐terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His‐TtCbs1 and His‐TtCsa1 were expressed and purified in vitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high Kₘ for O‐acetylserine and low Kₘ for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.

Network analysis is a systems biology-oriented approach based on graph theory that has been recently adopted in various fields of life sciences. Starting from mitochondrial proteomes purified from roots of Cucumis sativus plants grown under single or combined iron (Fe) and molybdenum (Mo) starvation, we reconstructed and analyzed at the topological level the protein–protein interaction (PPI) and co-expression networks. Besides formate dehydrogenase (FDH), already known to be involved in Fe and Mo nutrition, other potential mitochondrial hubs of Fe and Mo homeostasis could be identified, such as the voltage-dependent anion channel VDAC4, the beta-cyanoalanine synthase/cysteine synthase CYSC1, the aldehyde dehydrogenase ALDH2B7, and the fumaryl acetoacetate hydrolase. Network topological analysis, applied to plant proteomes profiled in different single or combined nutritional conditions, can therefore assist in identifying novel players involved in multiple homeostatic interactions.

Serine synthase (SS) from

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an

In a previous study, we found that H2S alleviates salinity stress in cucumber by maintaining the Na+/K+ balance and by regulating H2S metabolism and the oxidative stress response. However, little is known about the molecular mechanisms behind H2S-regulated salt-stress tolerance in cucumber. Here, an integrated transcriptomic and proteomic analysis based on RNA-seq and 2-DE was used to investigate the global mechanism underlying H2S-regulated salt-stress tolerance. In total, 11,761 differentially expressed genes (DEGs) and 61 differentially expressed proteins (DEPs) were identified. Analysis of the pathways associated with the DEGs showed that salt stress enriched expression of genes in primary and energy metabolism, such as photosynthesis, carbon metabolism and biosynthesis of amino acids. Application of H2S significantly decreased these DEGs but enriched DEGs related to plant-pathogen interaction, sulfur-containing metabolism, cell defense, and signal transduction pathways. Notably, changes related to sulfur-containing metabolism and cell defense were also observed through proteome analysis, such as Cysteine synthase 1, Glutathione S-transferase U25-like, Protein disulfide-isomerase, and Peroxidase 2. We present the first global analysis of the mechanism underlying H2S regulation of salt-stress tolerance in cucumber through tracking changes in the expression of specific proteins and genes.

Four different methods were evaluated to extract proteins from "Musang King" durian pulps and subsequently proteins with different abundance between fresh and long term frozen storage were identified using two-dimensional polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analyses. The acetone-phenol method was found to produce good protein yields and gave the highest gel resolution and reproducibility. Differential protein analyses of the durian pulp revealed that 15 proteins were down-regulated and three other proteins were up-regulated after a year of frozen storage. Isoflavone reductase-like protein, S-adenosyl methionine synthase, and cysteine synthase isoform were up-regulated during frozen storage. The down-regulation of proteins in frozen durian pulps indicated that frozen storage has affected proteins in many ways, especially in their functions related to carbohydrate and energy metabolisms, cellular components, and transport processes. This study will enable future detailed investigations of proteins associated with quality attributes of durians to be studied.

Salt stress is a major constrain to the productivity of nutritionally rich pigeonpea, an important legume of SE Asia and other parts of the world. The present study provides a comprehensive insight on integrated proteomic and transcriptomic analysis of root and shoot tissues of contrasting pigeonpea varieties (ICP1071- salt-sensitive; ICP7- salt-tolerant) to unravel salt stress induced pathways. Proteome analysis revealed 82 differentially expressed proteins (DEPs) with ≥±1.5 fold expression on 2-Dimensional (2D) gel. Of these, 25 DEPs identified through MALDI-TOF/TOF were classified using Uniprot software into functional categories. Pathways analyses using KAAS server showed the highest abundance of functional genes regulating metabolisms of carbohydrate followed by protein folding/degradation, amino acids and lipids. Expression studies on six genes (triosephosphate isomerase, oxygen evolving enhancer protein 1, phosphoribulokinase, cysteine synthase, oxygen evolving enhancer protein 2 and early nodulin like protein 2) with ≥±3 fold change were performed, and five of these showed consistency in transcript and protein expressions. Transcript analysis of root and shoot led to positive identification of 25 differentially expressed salt-responsive genes, with seven genes having ≥±5 fold change have diverse biological functions. Our combinatorial analysis suggests important role of these genes/proteins in providing salt tolerance in pigeonpea.

O-acetyl serine sulfhydrylase (OASS), referred to as cysteine synthase (CS), synthesizes cysteine from O-acetyl serine (OAS) and sulfur in bacteria and plants. The inherent challenge for CS is to overcome 4 to 6 log-folds stronger affinity for its natural inhibitor, serine acetyltransferase (SAT), as compared with its affinity for substrate, OAS. Our recent study showed that CS employs a novel competitive-allosteric mechanism to selectively recruit its substrate in the presence of natural inhibitor. In this study, we trace the molecular features that control selective substrate recruitment. To generalize our findings, we used CS from three different bacteria (Haemophilus, Salmonella, and Mycobacterium) as our model systems and analyzed structural and substrate-binding features of wild-type CS and its ∼13 mutants. Results show that CS uses a noncatalytic residue, M120, located 20 A away from the reaction center, to discriminate in favor of substrate. M120A and background mutants display significantly reduced substrate binding, catalytic efficiency, and inhibitor binding. Results shows that M120 favors the substrate binding by selectively enhancing the affinity for the substrate and disengaging the inhibitor by 20 to 286 and 5- to 3-folds, respectively. Together, M120 confers a net discriminative force in favor of substrate by 100- to 858-folds.

Food spoilage by certain species of bacteria is reported to be regulated by quorum sensing (QS). Acinetobacter johnsonii and Pseudomonas fluorescens, the major specific spoilage organisms, are found to be limited in their QS and co-culture interactions. The aim of this study was to determine how QS-regulated proteins affect the spoilage potential of co-cultured A. johnsonii and P. fluorescens obtained from spoiled bigeye tuna (Thunnus obesus) using a proteomics approach. The A. johnsonii, P. fluorescens, and their co-culture tested the N-acyl-homoserine lactone (AHL) activities using reporter Chromobacterium violaceum CV026 and LC-MS/MS in qualitative and quantitative approaches, respectively. These latter showed that, of the 470 proteins and 444 proteins in A. johnsonii (A) and P. fluorescens (P), respectively, 80 were significantly up-regulated and 97 were significantly down-regulated in A vs. AP, whereas 90 were up-regulated and 65 were down-regulated in P vs. AP. The differentially expressed proteins included the AI-2E family transporter OS, 50S ribosomal protein L3, thioredoxin reductase OS, cysteine synthase CysM OS, DNA-binding response regulator, and amino acid ABC transporter ATPase OS. The cellular process (GO:0009987), metabolic process (GO:0008152), and single-organism process (GO:0044699) were classified into the gene ontology (GO) term. In addition, energy production and conversion, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, post-translational modification, protein turnover, and chaperones were distributed into the clusters of orthologous groups of proteins (COG) terms. The KEGG pathways revealed that 84 and 77 differentially expressed proteins were divided into 20 KEGG pathways in A vs. AP and P vs. AP, respectively, and amino acid metabolism, carbohydrate metabolism, energy metabolism, and translation were significantly enriched. Proteins that correlated with the spoilage-related metabolic pathways, including thioredoxin reductase OS, cysteine synthase OS, and pyridoxal phosphate-dependent enzyme family protein OS, were identified. AI-2E family transporter OS and LuxR family transcriptional regulator OS were identified that related to the QS system. These findings provide a differential proteomic profile of co-culture in A. johnsonii and P. fluorescens, and have potential applications in QS and the regulation of spoilage potential.

Parental exposure to pathogens can prime offspring immunity in diverse organisms. The mechanisms by which this heritable priming occurs are largely unknown. Here we report that the soil bacteria Pseudomonas vranovensis is a natural pathogen of the nematode Caenorhabditis elegans and that parental exposure of animals to P. vranovensis promotes offspring resistance to infection. Furthermore, we demonstrate a multigenerational enhancement of progeny survival when three consecutive generations of animals are exposed to P. vranovensis. By investigating the mechanisms by which animals heritably adapt to P. vranovensis infection, we found that parental infection by P. vranovensis results in increased expression of the cysteine synthases cysl-1 and cysl-2 and the regulator of hypoxia inducible factor rhy-1 in progeny, and that these three genes are required for adaptation to P. vranovensis. These observations establish a CYSL-1, CYSL-2, and RHY-1 dependent mechanism by which animals heritably adapt to infection., Caenorhabditis elegans exhibits multigenerational adaptation to bacterial infection but the mechanisms remain unclear. Here, the authors show that C. elegans parental exposure to Pseudomonas vranovensis promotes offspring resistance to infection, a process mediated by the cysteine synthases CYSL-1 and CYSL-2.

To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery. ispartof: PLANT CELL AND ENVIRONMENT vol:43 issue:9 pages:2254-2271 ispartof: location:United States status: published

The lack of efficacy of current antibacterials to treat multidrug resistant bacteria poses a life-threatening alarm. In order to develop enhancers of the antibacterial activity, we carried out a medicinal chemistry campaign aiming to develop inhibitors of enzymes that synthesise cysteine and belong to the reductive sulphur assimilation pathway, absent in mammals. Previous studies have provided a novel series of inhibitors for O-acetylsulfhydrylase – a key enzyme involved in cysteine biosynthesis. Despite displaying nanomolar affinity, the most active representative of the series was not able to interfere with bacterial growth, likely due to poor permeability. Therefore, we rationally modified the structure of the hit compound with the aim of promoting their passage through the outer cell membrane porins. The new series was evaluated on the recombinant enzyme from Salmonella enterica serovar Typhimurium, with several compounds able to keep nanomolar binding affinity despite the extent of chemical manipulation.

Cysteine synthase A (CysK) catalyzes the last reaction of l-cysteine synthesis in bacteria, but its moonlighting functions have been revealed recently. In this study, CysK was overexpressed in Corynebacterium glutamicum IWJ001, an l-isoleucine producer. Compared with the control IWJ001/pDXW-8, IWJ001/pDXW-8-cysK cells grew fast during log phase, and produced 26.5% more l-isoleucine in flask fermentation and 23.5% more l-isoleucine in fed-batch fermentation. The key genes aspC, lysC, hom, thrB, ilvA, and ilvBN involved in l-isoleucine biosynthesis were all upregulated in IWJ001/pDXW-8-cysK, compared with IWJ001/pDXW-8. In addition, IWJ001/pDXW-8-cysK cells were longer and thicker than IWJ001/pDXW-8 cells. Compared with IWJ001/pDXW-8, the membrane permeability increased 15.8% and biofilm formation ability decreased 71.3% for IWJ001/pDXW-8-cysK cells. The results demonstrate that CysK overexpression in C. glutamicum is a good approach to enhance l-isoleucine production.

Increasing rates of drug-resistant Gram-negative (GN) infections, combined with a lack of new GN-effective antibiotic classes, are driving the need for the discovery of new agents. Bacterial metabolism represents an underutilized mechanism of action in current antimicrobial therapies. Therefore, we sought to identify novel antimetabolites that disrupt key metabolic pathways and explore the specific impacts of these agents on bacterial metabolism. This study describes the successful application of this approach to discover a new series of chemical probes, N-(phenyl)thioacetamide-linked 1,2,3-triazoles (TAT), that target cysteine synthase A (CysK), an enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to folate biosynthesis. TAT analog synthesis demonstrated a clear structure-activity relationship, and activity was confirmed against GN antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O-acetyl-l-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these antimetabolites impose substantial disruption of key metabolic networks beyond cysteine biosynthesis. This study highlights the potential of antimetabolite drug discovery as a promising approach to the discovery of novel GN antibiotics and the pharmacological promise of TAT CysK probes.

The l -cysteine is crucial for growth, survival, defense against oxidative stress, and pathogenesis of Entamoeba histolytica. The de novo biosynthesis of l -cysteine in E. histolytica, has a two-step pathway, where O-acetylserine sulfhydrylase (OASS) catalyses the last step by converting OAS to l -cysteine. This pathway is absent in humans and hence represents a promising target for novel therapeutics. E. histolytica expresses three isoforms of OASS and knockdown studies showed the importance of these enzymes for the survival of the pathogen. Here, we report the crystal structure of OASS isoform 3 from E. histolytica to 1.54 A resolution. The active site geometries and kinetics of EhOASS3 and EhOASS1 structures were found to be very similar. Small-molecule libraries were screened against EhOASS3 and compounds were shortlisted based on the docking scores. F3226-1387 showed best inhibition with IC50 of 38 μM against EhOASS3 and was able to inhibit the growth of the organism to 72%.

Garlic is a valuable source of healthy compounds, including secondary metabolites rich in sulphur such as cysteine sulphoxides (CSOs). Here, we present new qRT-PCR assays analysing the transcription of two genes encoding key enzymes in CSO biosynthetic pathways (cysteine synthase and alliinase) in developing garlic. We also identified a set of genes (ACT I, GAPDH, and TUB) to use as transcription normalisation controls. We showed that the (normalised) transcription of both enzymes was highest during sprouting and decreased significantly in fully developed leaves, which are the major CSO-producing organs. Transcriptional activity further declined at the end of the growing season. Different cultivars show similar sulphur metabolism gene expression when European garlics were compared to Chinese and American genotypes. The qRT-PCR assays presented are also suitable for investigating the effects of agricultural practices on CSO formation in garlic to satisfy consumer demands.

Large yellow croaker (Pseudosciaena crocea) is a popular and nutritious but also highly perishable fish species, with Shewanella baltica being the primary spoilage bacteria during low-temperature storage. Clarifying the factors promoting spoilage will facilitate efforts to predict and control the shelf life of foods. This study focused on spoilage-related genes in two Shewanella baltica strains with different spoilage potentials. Using whole genome sequencing and alignment, three distinguishing genes (torT, cysM and trxB) were identified. Further protein sequence comparison and protein structure modeling revealed possible motifs responsible for the spoilage activity. Moreover, diketopiperazine (DKP) quorum sensing (QS) signaling molecules regulated biofilm formation and spoilage gene expression, indicating a relationship between the QS system, biofilm formation and spoilage potential. Our results suggest that DKPs and spoilage genes are potential targets for developing novel food antiseptics, as well as new markers for fish product spoilage.

In Arabidopsis thaliana, cyanide is produced concomitantly with ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. In roots, CAS-C1 activity is essential to maintain a low level of cyanide for proper root hair development. Root hair elongation relies on polarized cell expansion at the growing tip, and we have observed that CAS-C1 locates in mitochondria and accumulates in root hair tips during root hair elongation, as shown by observing the fluorescence in plants transformed with the translational construct ProC1:CASC1-GFP, containing the complete CAS-C1 gene fused to GFP. Mutants in the SUPERCENTIPEDE (SCN1) gene, that regulate the NADPH oxidase RHD2/AtrbohC, are affected at the very early steps of the development of root hair that do not elongate and do not show a preferential localization of the GFP accumulation in the tips of the root hair primordia. Root hairs of mutants in CAS-C1 or RHD2/AtrbohC, which catalyzes the generation of ROS and the Ca2+ gradient, correctly start to grow out but they do not elongate either. Genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants were performed and the detail phenotypic and molecular characterization of the double mutants demonstrate that scn1 mutation is epistatic to cas-c1 and cas-c1 is epistatic to rhd2 mutation, indicating that CAS-C1 acts in early steps of the root hair development process. Moreover, our results show that the role of CAS-C1 in root hair elongation is independent of H2O2 production and of a direct NADPH oxidase inhibition by cyanide

Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).

Grass pea (Lathyrus sativus L.) is a worldwide popular pulse crop especially for its protein rich seeds with least production cost. However, the use of the crop became controversial due to the presence of non-protein amino acid, β-N-oxalyl-L-α, β-diaminopropionic acid (β-ODAP) in its seed and leaf, which is known as the principle neurotoxin to cause neurolathyrism (a motor neurodegenerative disease of humans and animals) during prolonged consumption as regular diet. Till date, the knowledge on β-ODAP biosynthesis in Lathyrus sp. is limited only to a small part of the complex bio-chemical steps involved including a few known sulfur-containing enzymes (viz. cysteine synthase, ODAP synthase etc.). In Lathyrus sativus, biosynthesis of β-ODAP varies differentially in a tissue-specific manner as well as in response to several environmental stresses viz. zinc deficiency, iron over-exposure, moisture stress etc. In the present study, a novel cysteine synthase gene (LsCSase) from Lathyrus sativus L was identified and characterized through bioinformatics approaches. The bioinformatic analysis revealed that LsCSase showed maximum similarity with the O-acetyl serine (thiol) lyase of Medicago truncatula with respect to several significant sequence-specific conserved motifs (cysK, CBS like, ADH_zinc_N, PALP), sub-cellular localization (chloroplast or cytoplasm) etc., similar to other members of cysteine synthase protein family. Moreover, the tissue-specific regulation of the LsCSase as well as its transcriptional activation under certain previously reported stressed conditions (low Zn+2-high Fe+2, PEG induced osmotic stress) were also documented through quantitative real-time PCR analyses, suggesting a possible link between the LsCSase gene activation and β-ODAP biosynthesis to manage external stresses in grass pea. This preliminary study offers a probable way towards the development of less toxic consumer-safe grass pea by down-regulation or deactivation of such gene/s (cysteine synthase) through genetic manipulations.