O-acetylserine(thiol)lyase Isoform Research Articles

Molecular cloning of putative chloroplastic cysteine synthase in Leucaena leucocephala.

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

A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms

An enzyme, O-acetylserine(thiol)lyase (OASTL), also known as O-acetylserine sulfhydrylase or cysteine synthase (CSase), catalyses the incorporation of sulfide into O-acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from Spinacia oleracea, (SoCSaseLP), but a recombinant SoCSaseLP produced in Escherichia coli did not show OASTL activity. The exon-intron structure of the SoCSaseLP gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a Beta vulgaris homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the SoCSaseLP gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione S-transferase (GST)-SoCSaseLP fusion protein was expressed in E. coli together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.

The role of compartment-specific cysteine synthesis for sulfur homeostasis during H2S exposure in Arabidopsis.

Sulfide is the end-product of assimilatory sulfate reduction in chloroplasts. It is then used by O-acetylserine(thiol)lyase (OAS-TL) to produce cysteine, the source of reduced sulfur in plants. While its formation in chloroplasts is essential for plant metabolism, sulfide is also a potent toxin mainly targeting respiration in mitochondria. Here, the application of sublethal concentrations of sulfide to Arabidopsis thaliana was used to by-pass assimilatory sulfate reduction, resulting in down-regulation of most genes of the pathway. The dualism of sulfide as substrate and toxin was investigated using knock-out mutants of the chloroplast-, mitochondrion- and cytosol-targeted OAS-TL isoforms. Surprisingly, growth retardation due to intoxication by sulfide was independent of the presence or absence of the three OAS-TL isoforms, indicating rapid exchange towards sulfur homoeostasis between the compartments. Cysteine, glutathione and sulfate, and less so S-sulfocysteine, were identified as major sinks for excess sulfide in wild-type plants. Additionally, the concentration of thiosulfate increased 1,000-fold, pointing towards a significant function of thiosulfate formation during H2S exposure. Synthesis of cysteine in the cytosol was found to be particularly important for accumulation of sulfite, sulfate and thiosulfate, indicating an important role for cytosolic OAS-TL for the re-oxidation of sulfide. The results show that thiosulfate and sulfate accumulation is strongly linked to cytosolic cysteine synthesis and that scavenging of sulfide by cysteine synthesis enhances sulfur compound accumulation. However, lack of cysteine synthesis in a subcellular compartment has no crucial consequences for toxicity and subsequent growth retardation.

Affinity Purification of O-Acetylserine(thiol)lyase from Chlorella sorokiniana by Recombinant Proteins from Arabidopsis thaliana.

In the unicellular green alga Chlorella sorokiniana (211/8 k), the protein O-acetylserine(thiol)lyase (OASTL), representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S) deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h) cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32–34 kDa and are thus slightly larger compared to those found in other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species.

An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase.

In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the Km was calculated to be 1,850±414 μM sulfide and the Vmax was 200.6±19.92 μM cysteine min(-1). The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.

Differential Response of Cysteine-deficient Lentil (<i>Lens culinaris</i> Medik.) Mutants Impaired in Foliar O-acetylserine(thiol)-lyase Expression

Lentil is a cool-season pulse crop, rich in protein but deficient in two sulphur-containing amino acids cysteine and methionine. Due to low genetic variability in existing germplasm, induced mutagenic technique has been adopted in lentil, and two mutant lines exhibiting poor growth and low dry weight were isolated in M 2 -mutagenized (0.10% and 0.15% EMS, 6 h) population of variety L 414. Further analysis revealed that plants from both mutant lines were highly deficient in seed cysteine (Cys) content, and thus, were tentatively designated as cysLc1 and cysLc2 mutants. Mutant plants were advanced to M 3 generation. Biochemical analysis through cysteine synthesizing pathway in leaves revealed that activity of serine acetyl transferase (SAT) was normal in both the mutant progenies but both were highly deficient in foliar O -acetylserine(thiol)-lyase (OAS-TL) activity. Transcriptomic analysis by qRT-PCR confirmed normal expression of SAT in both mutants but revealed differential expressions of two OAS-TL isoforms; OAS-TL 1 isoform was not detectable in cysLc1 mutant while expression of OAS-TL 2 isoform was totally repressed in leaves of cysLc2 mutant. Genetic studies and test of allelism pointed out that both the mutants were recessive and were complementing with each other to produce normal in F 1 and normal along with mutant plants in F 2 progeny. The progeny plants exhibiting normal phenotype showed normal mRNA transcripts of both OAS-TL isoforms. Being stable and self-fertile, the mutants will give vital clues in genetic basis of thiol-metabolic network of lentil crops.

Evidence for Several Cysteine Transport Mechanisms in the Mitochondrial Membranes of Arabidopsis thaliana

Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in the cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that exogenouslly supplied [(35)S]cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for in organello protein synthesis. Analysis of cysteine uptake by isolated mitochondria and mitoplasts indicates that cysteine is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In addition, cysteine uptake is dependent mainly on the ΔpH across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites in the cyanide detoxification-linked sulfide oxidation, but not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the transport of cysteine plays a pivotal role in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism.

Successful Fertilization Requires the Presence of at Least One Major O-Acetylserine(thiol)lyase for Cysteine Synthesis in Pollen of Arabidopsis

The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant.

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.

Cysteine homeostasis plays an essential role in plant immunity

• Cysteine is the metabolic precursor of essential biomolecules such as vitamins, cofactors, antioxidants and many defense compounds. The last step of cysteine metabolism is catalysed by O-acetylserine(thiol)lyase (OASTL), which incorporates reduced sulfur into O-acetylserine to produce cysteine. In Arabidopsis thaliana, the main OASTL isoform OAS-A1 and the cytosolic desulfhydrase DES1, which degrades cysteine, contribute to the cytosolic cysteine homeostasis. • Meta-analysis of the transcriptomes of knockout plants for OAS-A1 and for DES1 show a high correlation with the biotic stress series in both cases. • The study of the response of knockout mutants to plant pathogens shows that des1 mutants behave as constitutive systemic acquired resistance mutants, with high resistance to biotrophic and necrotrophic pathogens, salicylic acid accumulation and WRKY54 and PR1 induction, while oas-a1 knockout mutants are more sensitive to biotrophic and necrotrophic pathogens. However, oas-a1 knockout mutants lack the hypersensitive response associated with the effector-triggered immunity elicited by Pseudomonas syringae pv.tomato DC3000 avrRpm1. • Our results highlight the role of cysteine as a crucial metabolite in the plant immune response.

Enzymes of cysteine synthesis show extensive and conserved modifications patterns that include Nα-terminal acetylation

Biosynthesis of cysteine is a two-step process in higher plants subsequently catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which are present in cytosol, plastids and mitochondria. Recently, the distribution of SAT and OAS-TL in these subcellular compartments was shown to be crucial for efficient cysteine synthesis in Arabidopsis thaliana. In this study, the abundances of OAS-TLs were quantified independently by immunological detection in crude protein extracts and by SAT affinity purification (SAP) of OAS-TL. OAS-TL A and B were evidenced to be the most abundant isoforms in all analyzed tissues, which is consistent with micro array-based transcript analyses. Application of SAP to Arabidopsis revealed significant modification of the major OAS-TL isoforms present in cytosol, plastids and mitochondria into up to seven subspecies. Specific OAS-TL isoforms were found to be differentially modified in the leaves, roots, stem and cell culture. Sulphur deficiency did not alter modification of OAS-TL proteins purified from cell culture that showed the highest complexity of OAS-TL modifications. However, the pattern of OAS-TL modification was found to be stable within an analyzed tissue, pointing not only to a high reproducibility of SAP but likely biological significance of each subspecies. The most abundant OAS-TL subspecies in cytosol and plastids were subject of N-terminal processing followed by acetylation of the newly originated N-terminus. The mode of N(α)-terminal acetylation of OAS-TL and its possible biological function are discussed.

A mutation in the cytosolic O-acetylserine (thiol) lyase induces a genome-dependent early leaf death phenotype in Arabidopsis

BackgroundCysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol) lyase (OAS-TL) catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified.ResultsThe seedling lethal phenotype of the Arabidopsis onset of leaf death3-1 (old3-1) mutant is due to a point mutation in the OAS-A1 gene, encoding the cytosolic OAS-TL. The mutation causes a single amino acid substitution from Gly162 to Glu162, abolishing old3-1 OAS-TL activity in vitro. The old3-1 mutation segregates as a monogenic semi-dominant trait when backcrossed to its wild type accession Landsberg erecta (Ler-0) and the Di-2 accession. Consistent with its semi-dominant behaviour, wild type Ler-0 plants transformed with the mutated old3-1 gene, displayed the early leaf death phenotype. However, the old3-1 mutation segregates in an 11:4:1 (wild type: semi-dominant: mutant) ratio when backcrossed to the Colombia-0 and Wassilewskija accessions. Thus, the early leaf death phenotype depends on two semi-dominant loci. The second locus that determines the old3-1 early leaf death phenotype is referred to as odd-ler (for old3 determinant in the Ler accession) and is located on chromosome 3. The early leaf death phenotype is temperature dependent and is associated with increased expression of defence-response and oxidative-stress marker genes. Independent of the presence of the odd-ler gene, OAS-A1 is involved in maintaining sulphur and thiol levels and is required for resistance against cadmium stress.ConclusionsThe cytosolic OAS-TL is involved in maintaining organic sulphur levels. The old3-1 mutation causes genome-dependent and independent phenotypes and uncovers a novel function for the mutated OAS-TL in cell death regulation.

An O-Acetylserine(thiol)lyase Homolog with l-Cysteine Desulfhydrase Activity Regulates Cysteine Homeostasis in Arabidopsis

Cysteine (Cys) occupies a central position in plant metabolism due to its biochemical functions. Arabidopsis (Arabidopsis thaliana) cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of Cys. Because they are localized in the cytosol, plastids, and mitochondria, this results in multiple subcellular Cys pools. Much progress has been made on the most abundant OASTL enzymes; however, information on the less abundant OASTL-like proteins has been scarce. To unequivocally establish the enzymatic reaction catalyzed by the minor cytosolic OASTL isoform CS-LIKE (for Cys synthase-like; At5g28030), we expressed this enzyme in bacteria and characterized the purified recombinant protein. Our results demonstrate that CS-LIKE catalyzes the desulfuration of L-Cys to sulfide plus ammonia and pyruvate. Thus, CS-LIKE is a novel L-Cys desulfhydrase (EC 4.4.1.1), and we propose to designate it DES1. The impact and functionality of DES1 in Cys metabolism was revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. Mutation of the DES1 gene leads to premature leaf senescence, as demonstrated by the increased expression of senescence-associated genes and transcription factors. Also, the absence of DES1 significantly reduces the total Cys desulfuration activity in leaves, and there is a concomitant increase in the total Cys content. As a consequence, the expression levels of sulfur-responsive genes are deregulated, and the mutant plants show enhanced antioxidant defenses and tolerance to conditions that promote oxidative stress. Our results suggest that DES1 from Arabidopsis is an L-Cys desulfhydrase involved in maintaining Cys homeostasis, mainly at late developmental stages or under environmental perturbations.

Knocking Out Cytosolic Cysteine Synthesis Compromises the Antioxidant Capacity of the Cytosol to Maintain Discrete Concentrations of Hydrogen Peroxide in Arabidopsis

Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in cysteine (Cys) biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidopsis (Arabidopsis thaliana). Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favor of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to cadmium. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under nonstress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. Evidences that the mutation caused a perturbation in H2O2 homeostasis are that, in the knockout, H2O2 production was localized in shoots and roots; spontaneous cell death lesions occurred in the leaves; and lignification and guaiacol peroxidase activity were significantly increased. All these findings indicate that a deficiency of OAS-A1 in the cytosol promotes a perturbation in H2O2 homeostasis and that Cys is an important determinant of the antioxidative capacity of the cytosol in Arabidopsis.

Analysis of theArabidopsis O-Acetylserine(thiol)lyase Gene Family Demonstrates Compartment-Specific Differences in the Regulation of Cysteine Synthesis

Cys synthesis in plants takes place in plastids, cytosol, and mitochondria. Why Cys synthesis is required in all compartments with autonomous protein biosynthesis and whether Cys is exchanged between them has remained enigmatic. This question was addressed using Arabidopsis thaliana T-DNA insertion lines deficient in the final step of Cys biosynthesis catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL). Null alleles of oastlA or oastlB alone showed that cytosolic OAS-TL A and plastid OAS-TL B were completely dispensable, although together they contributed 95% of total OAS-TL activity. An oastlAB double mutant, relying solely on mitochondrial OAS-TL C for Cys synthesis, showed 25% growth retardation. Although OAS-TL C alone was sufficient for full development, oastlC plants also showed retarded growth. Targeted affinity purification identified the major OAS-TL-like proteins. Two-dimensional gel electrophoresis and mass spectrometry showed no compensatory changes of OAS-TL isoforms in the four mutants. Steady state concentrations of Cys and glutathione and pulse-chase labeling with [35S]sulfate indicated strong perturbation of primary sulfur metabolism. These data demonstrate that Cys and also sulfide must be sufficiently exchangeable between cytosol and organelles. Despite partial redundancy, the mitochondria and not the plastids play the most important role for Cys synthesis in Arabidopsis.

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.

O-acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana.

The synthesis of cysteine is positioned at a decisive stage of assimilatory sulphate reduction, marking the fixation of inorganic sulphide into a carbon skeleton. O-acetylserine (thiol) lyase (OAS-TL) catalyses the reaction of inorganic sulphide with O-acetylserine (OAS). Despite its prominent position in the pathway OAS-TL is generally regarded as a non-limiting enzyme without regulatory function, due to low substrate affinities and semi-constitutive expression patterns. To resolve this apparent contradiction, the kinetic properties of three OAS-TLs from Arabidopsis thaliana, localized in the cytosol (A), plastids (B), and mitochondria (C), were analysed. The recombinant expressed OAS-TLs were purified to apparent homogeneity without any fusion tag to maintain their native forms. The proteins displayed high specific activities of 550-900 micromol min(-1) mg(-1). Using an improved and highly sensitive assay method for cysteine determination, the apparent K(m)(sulphide) was 3-6 microM for OAS-TL A, B, and C and thus 10-100 times lower than previously reported for plant OAS-TLs. K(m)(OAS) was between 310 microM and 690 microM for OAS-TL isoform A, B, and C, whereas the apparent dissociation binding constant for OAS was much lower (K(d)<1 microM OAS). A HPLC method was developed for OAS quantification that revealed fast increases of the cellular OAS concentration in response to sulphate deprivation. The observed fluctuations of intracellular OAS concentrations, combined with the OAS dissociation constant and the catalytic properties of OAS-TL, support the model of a dynamic cysteine synthesis system with regulatory function as can be expected from the position of the reaction in the sulphur assimilation pathway.

Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes ( oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/ oasA2 and oasB/ oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and β-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein–protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.