O-acetylserine Research Articles

Structural Insights into Catalysis and Inhibition of O-Acetylserine Sulfhydrylase from Mycobacterium tuberculosis: CRYSTAL STRUCTURES OF THE ENZYME α-AMINOACRYLATE INTERMEDIATE AND AN ENZYME-INHIBITOR COMPLEX

Cysteine biosynthetic genes are up-regulated in the persistent phase of Mycobacterium tuberculosis, and the corresponding enzymes are therefore of interest as potential targets for novel antibacterial agents. cysK1 is one of these genes and has been annotated as coding for an O-acetylserine sulfhydrylase. Recombinant CysK1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine to cysteine. The crystal structure of the enzyme was determined to 1.8A resolution. CysK1 belongs to the family of fold type II PLP enzymes and is similar in structure to other O-acetylserine sulfhydrylases. We were able to trap the alpha-aminoacrylate reaction intermediate and determine its structure by cryocrystallography. Formation of the aminoacrylate complex is accompanied by a domain rotation resulting in active site closure. The aminoacrylate moiety is bound in the active site via the covalent linkage to the PLP cofactor and by hydrogen bonds of its carboxyl group to several enzyme residues. The catalytic lysine residue is positioned such that it can protonate the Calpha-carbon atom of the aminoacrylate only from the si-face, resulting in the formation of L-cysteine. CysK1 is competitively inhibited by a four-residue peptide derived from the C-terminal of serine acetyl transferase. The crystallographic analysis reveals that the peptide binds to the enzyme active site, suggesting that CysK1 forms an bi-enzyme complex with serine acetyl transferase, in a similar manner to other bacterial and plant O-acetylserine sulfhydrylases. The structure of the enzyme-peptide complex provides a framework for the design of strong binding inhibitors.

A newly discovered post-translational modification – the acetylation of serine and threonine residues

Recent studies on a bacterial virulence factor, YopJ of Yersinia, have led to the realization that the acetylation of serine and threonine residues could be an important form of post-translational modification in eukaryotes. Although the identification of the machinery used for the addition and removal of acetyl groups on serine or threonine residues is in its infancy, the enzymes thus-far studied provide early insight into the mechanism of this newly discovered post-translational modification, and hint at its potential importance. For example, acetylation can compete with phosphorylation targeted to the same residues and could, therefore, alter the course of signaling pathways. What are the implications for signal transduction in eukaryotes and how widespread could acetylation of serine and threonine prove to be?

Multiple fungal enzymes possess cysteine synthase activity in vitro

We present evidence that there are at least three Aspergillus nidulans enzymes which catalyze in vitro the reaction of O-acetylserine (OAS) with sulfide forming cysteine. This activity is shared by cysteine synthase (CS) encoded by the cysB gene, homocysteine synthase encoded by cysD and by at least one more enzyme. Moreover, arginine, histidine or proline starvation leads to derepression of CS activity even in the cysB,cysD double mutant strains, while neither cysB nor cysD gene transcription is derepressed by amino acid starvation. Using a cpcA mutant, we show that starvation-inducible CS activity is under control of cross-pathway regulation. We identify CysF as a putative CS in A. nidulans. However, cysF gene transcription is not elevated by amino acid starvation. Therefore, it seems that there exists yet another enzyme, thus far unidentified, which possesses CS activity. Using mutants impaired during various steps of cysteine synthesis we prove that the cysB-encoded enzyme is the only CS of physiological importance in the studied fungus. Similar results were obtained with Schizosaccharomyces pombe mutant strains impaired in cysteine synthesis, indicating that the presence of multiple enzymes with in vitro CS activity may be a common feature of many fungal species.

Association of Protein Biogenesis Factors at the Yeast Ribosomal Tunnel Exit Is Affected by the Translational Status and Nascent Polypeptide Sequence

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.

Dominant-Negative Modification Reveals the Regulatory Function of the Multimeric Cysteine Synthase Protein Complex in Transgenic Tobacco

Cys synthesis in plants constitutes the entry of reduced sulfur from assimilatory sulfate reduction into metabolism. The catalyzing enzymes serine acetyltransferase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL) reversibly form the heterooligomeric Cys synthase complex (CSC). Dominant-negative mutation of the CSC showed the crucial function for the regulation of Cys biosynthesis in vivo. An Arabidopsis thaliana SAT was overexpressed in the cytosol of transgenic tobacco (Nicotiana tabacum) plants in either enzymatically active or inactive forms that were both shown to interact efficiently with endogenous tobacco OAS-TL proteins. Active SAT expression resulted in a 40-fold increase in SAT activity and strong increases in the reaction intermediate OAS as well as Cys, glutathione, Met, and total sulfur contents. However, inactive SAT expression produced much greater enhancing effects, including 30-fold increased Cys levels, attributable, apparently, to the competition of inactive transgenic SAT with endogenous tobacco SAT for binding to OAS-TL. Expression levels of tobacco SAT and OAS-TL remained unaffected. Flux control coefficients suggested that the accumulation of OAS and Cys in both types of transgenic plants was accomplished by different mechanisms. These data provide evidence that the CSC and its subcellular compartmentation play a crucial role in the control of Cys biosynthesis, a unique function for a plant metabolic protein complex.

Yersinia Inhibits Host Signaling by Acetylating MAPK Kinases

Pathogenic Yersinia spp. secrete the effector YopJ (YopP) into host cells to counteract cytokine production and to induce programmed cell death (apoptosis). YopJ achieves these aims by inactivating mitogen-activated protein kinase (MAPK) and nuclear factor kappaB signaling pathways. YopJ was shown to bind to members of the MAPK kinase (MKK) family and was predicted to have protease activity toward ubiquitin (Ub)-like proteins. In a recent report, YopJ was demonstrated to inactivate MKKs via acetylation of critical serine or threonine residues. The ramifications of these exciting results are discussed in the context of other studies implicating YopJ as a Ub-like protease.

A ≥ 100000-MW Soybean Protein Fraction that Inhibits the Formation of Methanethiol and Hydrogen Sulfide in Aqueous Slurries of Isolated Soy Proteins with Added L-Cysteine

The addition of L-cysteine to aqueous slurries of commercial isolated soy proteins (ISP) increased methanethiol headspace levels by 17- to 36-fold over the control. Corresponding levels of hydrogen sulfide were about 10 to 19 times greater than methanethiol. Neither methanethiol nor hydrogen sulfide were detected when L-cysteine was added to aqueous slurries of hexane-defatted soy flour. The production of hydrogen sulfide and methanethiol in aqueous slurries of commercial ISP was inhibited by the addition of a component(s) recovered from the pH 4.6 supernatant obtained during laboratory preparation of ISP by isoelectric precipitation. The inhibitory component had a molecular weight (MW) of ≥ 100000 and an isoelectric point of about 5.9. This component was not serine acetyl transferase. Its inhibitory properties were inactivated at 70°C and diminished with elevated levels of methionine. Adding the ≥ 100000-MW soluble-proteins (from the isoelectric precipitation step) back to a nonheat-treated laboratory ISP during processing reduced the methanethiol level by 88%.

The effect of cadmium on sulfate assimilation gene expression in Arabidopsis thaliana

Plants deal with heavy metal stress by synthesizing peptides called phytochelatins. Phytochelatins are cysteine-rich peptides that chelate heavy metals such as cadmium. Cysteine is present in low concentrations in plants. It is likely that the synthesis of phytochelatins will increase the demand for cysteine. Our hypothesis is that metal tolerant plants are able to meet an increased demand for cysteine and wild type plants are not. The goal of our research is to find out how metal tolerant plants meet an increase in the need for cysteine imposed by cadmium. Is it by increasing the activity of the normal sulfate assimilation pathway or is there an alternate pathway by which cysteine is made available for phytochelatin production? Synthesis of cysteine begins when sulfate ions enter a cell using a transporter. Once inside, sulfate ion is adenylated in a reaction catalyzed by ATP sulfurylase (ATPS) converting it to adenosine 5′-phosphosulphate (APS). APS then goes through two more reactions catalyzed by the enzymes APS reductase (APSR) and sulfite reductase (SiR) to produce sulfide ion. Sulfide then reacts with O-acetylserine (OAS) to form cysteine. In this project we focused on the expression of ATPS, APSR and SiR in wild type Arabidopsis thaliana grown under various cadmium concentrations using reverse transcriptase PCR and real time PCR. This research is supported by the NIH, MBRS SCORE S06 GM52588.

Intracellular Bacteria Differentially Regulated Endothelial Cytokine Release by MAPK-Dependent Histone Modification

Epigenetic histone modifications contribute to the regulation of eukaryotic gene transcription. The role of epigenetic regulation in immunity to intracellular pathogens is poorly understood. We tested the hypothesis that epigenetic histone modifications influence cytokine expression by intracellular bacteria. Intracellular Listeria monocytogenes, but not noninvasive Listeria innocua, induced release of distinct CC and CXC chemokines, as well as Th1 and Th2 cytokines and growth factors by endothelial cells. Cytokine expression was in part dependent on p38 MAPK and MEK1. We analyzed global histone modification and modifications in detail at the gene promoter of IL-8, which depended on both kinase pathways, and of IFN-gamma, which was not blocked by kinase inhibition. Intracellular Listeria induced time-dependent acetylation (lysine 8) of histone H4 and phosphorylation/acetylation (serine 10/lysine 14) of histone H3 globally and at the il8 promoter in HUVEC, as well as recruitment of the histone acetylase CREB-binding protein. Inhibitors of p38 MAPK and MEK1 reduced lysine 8 acetylation of histone H4 and serine 10/lysine 14 phosphorylation/acetylation of histone H3 in Listeria-infected endothelial cells and disappearance of histone deacetylase 1 at the il8 promoter in HUVEC. In contrast, IFN-gamma gene transcription was activated by Listeria monocytogenes independent of p38 MAPK and MEK1, and histone phosphorylation/acetylation remained unchanged in infected cells at the IFN-gamma promoter. Specific inhibition of histone deacetylases by trichostatin A increased Listeria-induced expression of IL-8, but not of IFN-gamma, underlining the specific physiological impact of histone acetylation. In conclusion, MAPK-dependent epigenetic modifications differentially contributed to L. monocytogenes-induced cytokine expression by human endothelial cells.

N‐terminal acetylation and protonation of individual hemoglobin subunits: Position‐dependent effects on tetramer strength and cooperativity

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the alpha- or the beta-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the alpha-subunits of liganded hemoglobin results in a significantly increased basicity (increased pK(a) values) and a reduction in the strength of subunit interactions at the allosteric tetramer-dimer interface. Cooperativity in O(2) binding is also decreased. Substitution of Ala at the N-terminals of the beta-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer-dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the alpha-subunit, the slope of the plot of the tetramer-dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the alpha-subunits is less extensive than that of the beta-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403

Cysteine and methionine availability influences many processes in the cell. In bacteria, transcription of the specific genes involved in the synthesis of these two amino acids is usually regulated by different mechanisms or regulators. Pathways for the synthesis of cysteine and methionine and their interconversion were experimentally determined for Lactococcus lactis, a lactic acid bacterium commonly found in food. A new gene, yhcE, was shown to be involved in methionine recycling to cysteine. Surprisingly, 18 genes, representing almost all genes of these pathways, are under the control of a LysR-type activator, FhuR, also named CmbR. DNA microarray experiments showed that FhuR targets are restricted to this set of 18 genes clustered in seven transcriptional units, while cysteine starvation modifies the transcription level of several other genes potentially involved in oxidoreduction processes. Purified FhuR binds a 13-bp box centered 46 to 53 bp upstream of the transcriptional starts from the seven regulated promoters, while a second box with the same consensus is present upstream of the first binding box, separated by 8 to 10 bp. O-Acetyl serine increases FhuR binding affinity to its binding boxes. The overall view of sulfur amino acid metabolism and its regulation in L. lactis indicates that CysE could be a master enzyme controlling the activity of FhuR by providing its effector, while other controls at the enzymatic level appear to be necessary to compensate the absence of differential regulation of the genes involved in the interconversion of methionine and cysteine and other biosynthesis genes.

O-Acetylserine and the Regulation of Expression of Genes Encoding Components for Sulfate Uptake and Assimilation in Potato

cDNAs encoding a high-affinity sulfate transporter and an adenosine 5'-phosphosulfate reductase from potato (Solanum tuberosum L. cv Desiree) have been cloned and used to examine the hypothesis that sulfate uptake and assimilation is transcriptionally regulated and that this is mediated via intracellular O-acetylserine (OAS) pools. Gas chromotography coupled to mass spectrometry was used to quantify OAS and its derivative, N-acetylserine. Treatment with external OAS increased sulfate transporter and adenosine 5'-phosphosulfate reductase gene expression consistent with a model of transcriptional induction by OAS. To investigate this further, the Escherichia coli gene cysE (serine acetyltransferase EC 2.3.1.30), which synthesizes OAS, has been expressed in potato to modify internal metabolite pools. Transgenic lines, with increased cysteine and glutathione pools, particularly in the leaves, had increased sulfate transporter expression in the roots. However, the small increases in the OAS pools were not supportive of the hypothesis that this molecule is the signal of sulfur (S) nutritional status. In addition, although during S starvation the content of S-containing compounds decreased (consistent with derepression as a mechanism of regulation), OAS pools increased only following extended starvation, probably as a consequence of the S starvation. Taken together, expression of these genes may be induced by a demand-driven model, via a signal from the shoots, which is not OAS. Rather, the signal may be the depletion of intermediates of the sulfate assimilation pathway, such as sulfide, in the roots. Finally, sulfate transporter activity did not increase in parallel with transcript and protein abundance, indicating additional posttranslational regulatory mechanisms.

Mechanism of the Addition Half of the O-Acetylserine Sulfhydrylase-A Reaction

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the beta-acetoxy group of O-acetyl-l-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the alpha-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the alpha-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK(a) > 7, while the rate constant is pH-independent for substrates with a pK(a) < 7 suggesting that the pK(a)s of the substrate and an enzyme group are important in this half of the reaction. In D(2)O, at low pD values, the amino acid external Schiff base is trapped, while in H(2)O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new beta-substituted amino acids were produced and characterized by (1)H NMR spectroscopy.

Impact of Reduced O-Acetylserine(thiol)lyase Isoform Contents on Potato Plant Metabolism

Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H(2)S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.

Engineering Soybean for Enhanced Sulfur Amino Acid Content

Soybean [Glycine max (L.) Merr.] is an important protein source in human diets and animal rations. Additionally, human consumption of soybean protein is reported to provide specific health benefits. Since methionine and cysteine contents limit the nutritional value of soybean, efforts involving both traditional breeding and genetic engineering have been employed in attempts to increase the presence of these essential amino acids. Traditional breeding has been primarily utilized to increase the total protein content but not to enhance the sulfur amino acid content of soybean. Although mutagenesis in conjunction with traditional breeding is a viable approach for enhancing the sulfur amino acid content of soybean, genetic engineering appears to be a more realistic approach. Introduction of methionine‐rich heterologous proteins has resulted in a modest increase of this amino acid in soybean. Either elevating the expression of endogenous methionine‐rich proteins or introducing synthetic proteins containing a high percentage of essential amino acids are other possible approaches that may increase the nutritional quality of the seed. Even though considerable progress has been made in enhancing the methionine content of soybean, several obstacles remain. A thorough understanding the sulfur assimilatory pathway in soybean is a prerequisite for improving the sulfur amino acid content. Expression of feedback‐insensitive forms of serine acetyl transferase and O‐acetylserine (thiol) lyase, two key enzymes in sulfur assimilatory pathway, could lead to an increase in the availability of sulfur amino acids. An adequate supply of sulfur amino acids in developing seeds may facilitate accumulation of sulfur‐rich proteins to a level sufficient to meet the nutritional requirement of livestock and poultry.

Independent roles of glutathione and O-acetyl-L-serine in regulation of sulfur-responsive gene expression in Arabidopsis thaliana

We investigated the effects of a four-day treatment with 0.3 mM glutathione (GSH) and 10 mM O-acetyl-L- serine (OAS), negative and positive regulators of sulfur-responsive gene expression, respectively, alone or in combination, on the sulfur-responsive gene expression in ten-day-old Arabidopsis thaliana plants. We determined the relationship between the concentrations of GSH, OAS and sulfate, and expression of sulfur-responsive genes in rosette leaves. The concentrations of GSH and OAS correlated negatively and positively with expression of genes, respectively, suggesting that GSH and OAS independently affect expression of sulfur-responsive genes.

Chemical Mechanism of the Serine Acetyltransferase from Haemophilus influenzae

The pH dependence of kinetic parameters was determined in both reaction directions to obtain information about the acid-base chemical mechanism of serine acetyltransferase from Haemophilus influenzae (HiSAT). The maximum rates in both reaction directions, as well as the V/K(serine) and V/K(OAS), decrease at low pH, exhibiting a pK of approximately 7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V(1)/E(t), V(1)/K(serine)E(t), V/K(AcCoA)E(t), V(2)/E(t), V(2)/K(OAS)E(t), and V/K(CoA)E(t) are 3300 +/- 180 s(-1), (9.6 +/- 0.4) x 10(5) M(-1) s(-1), 3.3 x 10(6) M(-1) s(-1), 420 +/- 50 s(-1), (2.1 +/- 0.5) x 10(4) M(-1) s(-1), and (4.2 +/- 0.7) x 10(5) M(-1) s(-1), respectively. The K(i) values for the competitive inhibitors glycine and l-cysteine are pH-independent. The solvent deuterium kinetic isotope effects on V and V/K in the direction of serine acetylation are 1.9 +/- 0.2 and 2.5 +/- 0.4, respectively, and the proton inventories are linear for both parameters. Data are consistent with a single proton in flight in the rate-limiting transition state. A general base catalytic mechanism is proposed for the serine acetyltransferase. Once acetyl-CoA and l-serine are bound, an enzymic general base accepts a proton from the l-serine side chain hydroxyl as it undergoes a nucleophilic attack on the carbonyl of acetyl-CoA. The same enzyme residue then functions as a general acid, donating a proton to the sulfur atom of CoASH as the tetrahedral intermediate collapses, generating the products OAS and CoASH. The rate-limiting step in the reaction at limiting l-serine levels is likely formation of the tetrahedral intermediate between serine and acetyl-CoA.

Regulation of sulfate uptake and xylem loading of poplar roots (Populus tremula x P. alba)

Sulfate transport processes and its regulation were studied in roots of poplar trees (Populus tremula x P. alba). From the exponential increase in sulfate uptake with temperature an activation energy (Ea) of 9.0±0.8 kJ mol−1 was calculated. In the concentration range 0.005–10 mM sulfate uptake showed biphasic Michaelis-Menten kinetics with a Km of 3.2±3.4 μM and a Vmax of 49±11 nmol SO42− g−1 FW h−1 for the high-affinity uptake system (phase 1) and a Km of 1.33±0.41 mM and a Vmax of 255±25 nmol SO42− g−1 FW h−1 for the low-affinity system (phase 2). Xylem loading decreased linearly with temperature and remained unchanged within the sulfate concentration range studied. Regulation of sulfate uptake and xylem loading by O-acetyl serine (OAS), Cys, reduced glutathione (GSH), Met and S-methylmethionine (SMM) were tested by perfusion into the xylem sap with the pressure probe and by addition to the incubation medium. When added directly to the transport medium, Cys and GSH repressed, and OAS stimulated sulfate uptake; xylem loading was stimulated by Cys, repressed by GSH and only slightly affected by OAS. When perfused into the xylem, none of the compounds tested affected sulfate uptake of excised roots, but xylem loading was stimulated by SMM and OAS and repressed by Met. Apparently, the site of application strongly determined the effect of regulatory compounds of sulfate transport processes.

Role of CysE in production of an extracellular signaling molecule in Providencia stuartii and Escherichia coli: loss of CysE enhances biofilm formation in Escherichia coli.

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4- to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.

Increased glutathione biosynthesis plays a role in nickel tolerance in thlaspi nickel hyperaccumulators.

Worldwide more than 400 plant species are now known that hyperaccumulate various trace metals (Cd, Co, Cu, Mn, Ni, and Zn), metalloids (As) and nonmetals (Se) in their shoots. Of these, almost one-quarter are Brassicaceae family members, including numerous Thlaspi species that hyperaccumulate Ni up to 3% of there shoot dry weight. We observed that concentrations of glutathione, Cys, and O-acetyl-l-serine (OAS), in shoot tissue, are strongly correlated with the ability to hyperaccumulate Ni in various Thlaspi hyperaccumulators collected from serpentine soils, including Thlaspi goesingense, T. oxyceras, and T. rosulare, and nonaccumulator relatives, including T. perfoliatum, T. arvense, and Arabidopsis thaliana. Further analysis of the Austrian Ni hyperaccumulator T. goesingense revealed that the high concentrations of OAS, Cys, and GSH observed in this hyperaccumulator coincide with constitutively high activity of both serine acetyltransferase (SAT) and glutathione reductase. SAT catalyzes the acetylation of l-Ser to produce OAS, which acts as both a key positive regulator of sulfur assimilation and forms the carbon skeleton for Cys biosynthesis. These changes in Cys and GSH metabolism also coincide with the ability of T. goesingense to both hyperaccumulate Ni and resist its damaging oxidative effects. Overproduction of T. goesingense SAT in the nonaccumulator Brassicaceae family member Arabidopsis was found to cause accumulation of OAS, Cys, and glutathione, mimicking the biochemical changes observed in the Ni hyperaccumulators. In these transgenic Arabidopsis, glutathione concentrations strongly correlate with increased resistance to both the growth inhibitory and oxidative stress induced effects of Ni. Taken together, such evidence supports our conclusion that elevated GSH concentrations, driven by constitutively elevated SAT activity, are involved in conferring tolerance to Ni-induced oxidative stress in Thlaspi Ni hyperaccumulators.