Mitochondrial cysteine synthase complex regulates o-acetylserine biosynthesis in plants

Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids, and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial. Here, we demonstrate the interaction between mitochondrial SAT3 and OAS-TL C in planta by FRET and establish the role of the mitochondrial CSC in the regulation of cysteine synthesis. NMR spectroscopy of isolated mitochondria from WT, serat2;2, and oastl-C plants showed the SAT-dependent export of OAS. The presence of cysteine resulted in reduced OAS export in mitochondria of oastl-C mutants but not in WT mitochondria. This is in agreement with the stronger in vitro feedback inhibition of free SAT by cysteine compared with CSC-bound SAT and explains the high OAS export rate of WT mitochondria in the presence of cysteine. The predominant role of mitochondrial OAS synthesis was validated in planta by feeding [(3)H]serine to the WT and loss-of-function mutants for OAS-TLs in the cytosol, plastids, and mitochondria. On the basis of these results, we propose a new model in which the mitochondrial CSC acts as a sensor that regulates the level of SAT activity in response to sulfur supply and cysteine demand.     

Allosterically gated enzyme dynamics in the cysteine synthase complex regulate cysteine biosynthesis in Arabidopsis thaliana

Plants and bacteria assimilate sulfur into cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase complex (CSC), which consists of serine-acetyl-transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL) enzymes. The activity of OAS-TL is reduced by formation of the CSC. Although this reduction is an inherent part of the self-regulation cycle of cysteine biosynthesis, there has until now been no explanation as to how OAS-TL loses activity in plants. Complexation of SAT and OAS-TL involves binding of the C-terminal tail of SAT in one of the active sites of the homodimeric OAS-TL. We here explore the flexibility of the unoccupied active site in Arabidopsis thaliana cytosolic and mitochondrial OAS-TLs. Our results reveal two gates in the OAS-TL active site that define its accessibility. The observed dynamics of the gates show allosteric closure of the unoccupied active site of OAS-TL in the CSC, which can hinder substrate binding, abolishing its turnover to cysteine.     

Functional analysis of the cysteine synthase protein complex from plants: structural, biochemical and regulatory properties

Cysteine synthesis in plants represents the final step of assimilatory sulfate reduction and the almost exclusive entry reaction of reduced sulfur into metabolism not only of plants, but also the human food chain in general. It is accomplished by the sequential reaction of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they form the hetero-oligomeric cysteine synthase complex (CSC). Recent evidence is reviewed that identifies the dual function of the CSC as a sensor and as part of a regulatory circuit that controls cellular sulfur homeostasis. Computational modeling of three-dimensional structures of plant SAT and OAS-TL based on the crystal structure of the corresponding bacterial enzymes supports quaternary conformations of SAT as a dimer of trimers and OAS-TL as a homodimer. These findings suggest an overall alpha6beta4 structure of the subunits of the plant CSC. Kinetic measurements of CSC dissociation triggered by the reaction intermediate O-acetylserine as well as CSC stabilization by sulfide indicate quantitative reactions that are suited to fine-tune the equilibrium between free and associated CSC subunits. In addition, in vitro data show that SAT requires binding to OAS-TL for full activity, while at the same time bound OAS-TL becomes inactivated. Since OAS concentrations inside cells increase upon sulfate deficiency, whereas sulfide concentrations most likely decrease, these data suggest the dissociation of the CSC in vivo, accompanied by inactivation of SAT and activation of OAS-TL function in their free homo-oligomer states. Biochemical evidence describes this protein-interaction based mechanism as reversible, thus closing the regulatory circuit. The properties of the CSC and its subunits are therefore consistent with models of positive regulation of sulfate uptake and reduction in plants by OAS as well as a demand-driven repression/de-repression by a sulfur intermediate, such as sulfide.     

Three-stage assembly of the cysteine synthase complex from Escherichia coli

Control of sulfur metabolism in plants and bacteria is linked, in significant measure, to the behavior of the cysteine synthase complex (CSC). The complex is comprised of the two enzymes that catalyze the final steps in cysteine biosynthesis: serine O-acetyltransferase (SAT, EC 2.3.1.30), which produces O-acetyl-L-serine, and O-acetyl-L-serine sulfhydrylase (OASS, EC 2.5.1.47), which converts it to cysteine. SAT (a dimer of homotrimers) binds a maximum of two molecules of OASS (a dimer) in an interaction believed to involve docking of the C terminus from a protomer in an SAT trimer into an OASS active site. This interaction inactivates OASS catalysis and prevents further binding to the trimer; thus, the system exhibits a contact-induced inactivation of half of each biomolecule. To better understand the dynamics and energetics that underlie formation of the CSC, the interactions of OASS and SAT from Escherichia coli were studied at equilibrium and in the pre-steady state. Using an experimental strategy that initiates dissociation of the CSC at different points in the CSC-forming reaction, three stable forms of the complex were identified. Comparison of the binding behaviors of SAT and its C-terminal peptide supports a mechanism in which SAT interacts with OASS in a non-allosteric interaction involving its C terminus. This early docking event appears to fasten the proteins in close proximity and thus prepares the system to engage in a series of subsequent, energetically favorable isomerizations that inactivate OASS and produce the fully isomerized CSC.     

The relevance of compartmentation for cysteine synthesis in phototrophic organisms

In the vascular plant Arabidopsis thaliana, synthesis of cysteine and its precursors O-acetylserine and sulfide is distributed between the cytosol, chloroplasts, and mitochondria. This compartmentation contributes to regulation of cysteine synthesis. In contrast to Arabidopsis, cysteine synthesis is exclusively restricted to chloroplasts in the unicellular green alga Chlamydomonas reinhardtii. Thus, the question arises, whether specification of compartmentation was driven by multicellularity and specified organs and tissues. The moss Physcomitrella patens colonizes land but is still characterized by a simple morphology compared to vascular plants. It was therefore used as model organism to study evolution of compartmented cysteine synthesis. The presence of O-acetylserine(thiol)lyase (OAS-TL) proteins, which catalyze the final step of cysteine synthesis, in different compartments was applied as criterion. Purification and characterization of native OAS-TL proteins demonstrated the presence of five OAS-TL protein species encoded by two genes in Physcomitrella. At least one of the gene products is dual targeted to plastids and cytosol, as shown by combination of GFP fusion localization studies, purification of chloroplasts, and identification of N termini from native proteins. The bulk of OAS-TL protein is targeted to plastids, whereas there is no evidence for a mitochondrial OAS-TL isoform and only a minor part of OAS-TL protein is localized in the cytosol. This demonstrates that subcellular diversification of cysteine synthesis is already initialized in Physcomitrella but appears to gain relevance later during evolution of vascular plants.     

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.     

Crystal structure of serine acetyl transferase from Brucella abortus and its complex with coenzyme A

Brucella abortus is the major cause of premature foetal abortion in cattle, can be transmitted from cattle to humans, and is considered a powerful biological weapon. De novo cysteine biosynthesis is one of the essential pathways reported in bacteria, protozoa, and plants. Serine acetyltransferase (SAT) initiates this reaction by catalyzing the formation of O-acetylserine (OAS) using l-serine and acetyl coenzyme A as substrates. Here we report kinetic and crystallographic studies of this enzyme from B. abortus. The kinetic studies indicate that cysteine competitively inhibits the binding of serine to B. abortus SAT (BaSAT) and noncompetitively inhibits the binding of acetyl coenzyme A. The crystal structures of BaSAT in its apo state and in complex with coenzyme A (CoA) were determined to 1.96 Å and 1.87 Å resolution, respectively. BaSAT was observed as a trimer in a size exclusion column; however, it was seen as a hexamer in dynamic light scattering (DLS) studies and in the crystal structure, indicating it may exist in both states. The complex structure shows coenzyme A bound to the C-terminal region, making mostly hydrophobic contacts from the center of the active site extending up to the surface of the protein. There is no conformational difference in the enzyme between the apo and the complexed states, indicating lock and key binding and the absence of an induced fit mechanism.     

Cysteine synthesis in plants: protein-protein interactions of serine acetyltransferase from Arabidopsis thaliana

The biosynthesis of cysteine represents the final step of sulfate assimilation in bacteria and plants. It is catalyzed by the sequential action of serine acetyltransferase (SAT) and O -acetylserine (thiol) lyase (OAS-TL) which form a cysteine synthase (CS) complex in vitro . SAT and OAS-TL from Arabidopsis thaliana have previously been cloned, and now the first evidence is presented for the CS complex and SAT self-interaction in vivo employing the yeast two-hybrid system. Application of this method proved to be an efficient tool for the analysis of protein-protein interactions within a plant metabolic protein complex. Mapping of SAT domain structure revealed two new, independent domains with specific functions in protein-protein interaction. Analysis using truncated proteins proved the C-terminus of SAT to be sufficient for association with OAS-TL and to correlate with the putative transferase activity domain. SAT/SAT interaction was localized in the central region of the protein and occured also between SAT isoforms. Both protein interaction domains coincided with distinct α-helical and β-sheet clusters and together correlated with the minimal protein structure required for SAT catalysis as shown by functional complementation of an Escherichia coli mutant. The homo- and hetero-oligomerization properties are discussed with respect to the assumed function of the CS complex in metabolic channeling and activation of SAT by interaction with OAS-TL.     

A mechanistic model of the cysteine synthase complex

Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is composed of the enzymes serine acetyl transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL). Although it is experimentally known that formation of the CS complex influences cysteine production, the exact biological function of the CS complex, the mechanism of reciprocal regulation of the constituent enzymes and the structure of the complex are still poorly understood. Here, we used docking techniques to construct a model of the CS complex from mitochondrial Arabidopsis thaliana. The three-dimensional structures of the enzymes were modeled by comparative techniques. The C-termini of SAT, missing in the template structures but crucial for CS formation, were modeled de novo. Diffusional encounter complexes of SAT and OAS-TL were generated by rigid-body Brownian dynamics simulation. By incorporating experimental constraints during Brownian dynamics simulation, we identified complexes consistent with experiments. Selected encounter complexes were refined by molecular dynamics simulation to generate structures of bound complexes. We found that although a stoichiometric ratio of six OAS-TL dimers to one SAT hexamer in the CS complex is geometrically possible, binding energy calculations suggest that, consistent with experiments, a ratio of only two OAS-TL dimers to one SAT hexamer is more likely. Computational mutagenesis of residues in OAS-TL that are experimentally significant for CS formation hindered the association of the enzymes due to a less-favorable electrostatic binding free energy. Since the enzymes from A. thaliana were expressed in Escherichia coli, the cross-species binding of SAT and OAS-TL from E. coli and A. thaliana was explored. The results showed that reduced cysteine production might be due to a cross-binding of A. thaliana OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation.     

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.     

Tumor Cells Positive and Negative for the Common Cancer Stem Cell Markers Are Capable of Initiating Tumor Growth and Generating Both Progenies

The cancer stem cell (CSC) model depicts that tumors are hierarchically organized and maintained by CSCs lying at the apex. CSCs have been “identified” in a variety of tumors through the tumor-forming assay, in which tumor cells distinguished by a certain cell surface marker (known as a CSC marker) were separately transplanted into immunodeficient mice. In such assays, tumor cells positive but not negative for the CSC marker (hereby defined as CSC⁺ and CSC⁻ cells, respectively) have the ability of tumor-forming and generating both progenies. However, here we show that CSC⁺ and CSC⁻ cells exhibit similar proliferation in the native states. Using a cell tracing method, we demonstrate that CSC⁻ cells exhibit similar tumorigenesis and proliferation as CSC⁺ cells when they were co-transplanted into immunodeficient mice. Through serial single-cell derived subline construction, we further demonstrated that CSC⁺ and CSC⁻ cells from CSC marker expressing tumors could invariably generate both progenies, and their characteristics are maintained among different generations irrespective of the origins (CSC⁺-derived or CSC⁻-derived). These findings demonstrate that tumorigenic cells cannot be distinguished by common CSC markers alone and we propose that cautions should be taken when using these markers independently to identify cancer stem cells due to the phenotypic plasticity of tumor cells.     

The serine acetyltransferase gene family in Arabidopsis thaliana and the regulation of its expression by cadmium

Expression of the serine acetyltransferase (SAT) gene family from Arabidopsis thaliana was investigated in response to treatment with the heavy metal cadmium (Cd). A fourth member of the SAT gene family, Sat-106, was also cloned and the complete SAT gene family from A. thaliana is discussed. Northern analysis of the gene family revealed tissue-specific expression patterns for each isogene. A. thaliana plants grown under 50 M CdCl₂ for a 24 h time course were also used for northern analysis. Expression of all SAT genes was increased to some extent by Cd treatment. Sat-5 expression showed particularly high levels of induction in the leaves of treated plants and was chosen for study by in situ hybridisation. Sat-5 expression was induced in the root and stem cortex and the leaf lamella and trichomes in response to heavy metal stress. SAT and its product O-acetylserine have previously been shown to be implicated in the control of sulphate reduction and cysteine biosynthesis in plants. These results suggest that specific SAT isoforms have a role in increasing cysteine production under conditions of heavy-metal stress when increased biosynthesis of glutathione and phytochelatins is required for detoxification purposes.     

Pausing of Golgi Bodies on Microtubules Regulates Secretion of Cellulose Synthase Complexes in Arabidopsis

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by plasma membrane–bound complexes containing cellulose synthase proteins (CESAs). Here, we establish a role for the cytoskeleton in intracellular trafficking of cellulose synthase complexes (CSCs) through the in vivo study of the green fluorescent protein (GFP)-CESA3 fusion protein in Arabidopsis thaliana hypocotyls. GFP-CESA3 localizes to the plasma membrane, Golgi apparatus, a compartment identified by the VHA-a1 marker, and, surprisingly, a novel microtubule-associated cellulose synthase compartment (MASC) whose formation and movement depend on the dynamic cortical microtubule array. Osmotic stress or treatment with the cellulose synthesis inhibitor CGA 325''615 induces internalization of CSCs in MASCs, mimicking the intracellular distribution of CSCs in nongrowing cells. Our results indicate that cellulose synthesis is coordinated with growth status and regulated in part through CSC internalization. We find that CSC insertion in the plasma membrane is regulated by pauses of the Golgi apparatus along cortical microtubules. Our data support a model in which cortical microtubules not only guide the trajectories of CSCs in the plasma membrane, but also regulate the insertion and internalization of CSCs, thus allowing dynamic remodeling of CSC secretion during cell expansion and differentiation.     

The Role of Heme Binding by DNA-protective Protein from Starved Cells (Dps) in the Tolerance of Porphyromonas gingivalis to Heme Toxicity

The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent K_d of 3.7 × 10⁻⁸ m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H₂O₂-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria.     

Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants

Summary. Serine acetyltransferase (SATase) and cysteine synthase (O-acetylserine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by l-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by l-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for l-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by l-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F₁) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 μL L⁻¹ sulfur dioxide, both the cysteine and glutathione contents in leaves of F₁ plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants. Received November 27, 2001 Accepted December 21, 2001     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

Regulation of sulphur assimilation in lettuce plants in the presence of selenium

Selenium (Se) is considered an essential trace element for animals because of its nutritional and clinical value, including its special relevance in cancer prevention, and thus Se is at present used in biofortification programmes. However, possible effects of Se application on S metabolism and plant growth are still not clear. Thus, we analysed the effect that Se application in two different forms (selenate versus selenite) exerts on the S metabolism in lettuce plants grown for 66 days. Our results indicate that the application of selenite as opposed to selenate does not affect the foliar concentration of S. With respect to different enzymes in charge of sulphate (SO₄²⁻) assimilation, the ATP-sulphurylase activity varies only with the application of different rates of selenate, while the activity of O-acetylserine(thiol)lyase (OAS-TL) and serine-acetyltransferase (SAT) increase in activity mainly when selenite is applied. Finally, the concentration of cysteine (Cys) and total thiols (SH-total), fundamentally in the selenate treatments, increased with shoot biomass. In conclusion, this study confirms that the form and application rate of Se affects S assimilation, selenate being the more suitable form to improve effectiveness of the biofortification programme with this trace element.     

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E_m) at pH 7.0 of approximately +210, −340, and −400 mV for the His/Met, His/Cys⁻, and active site His/CysS⁻-ligated heme, respectively. Exposing SoxAX to S₂O₄²⁻, a substrate analog with E_m ∼−450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E_m ∼−1140 mV), allows cyanide to displace the cysteine persulfide (CysS⁻) ligand to the active site heme. This provides the first evidence for the dissociation of CysS⁻ that has been proposed as a key event in SoxAX catalysis.