Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast.     

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs.     

Immuno-gold Localization of Nitrate Reductase in Spinach (Spinacia oleracea) Leaves

The intracellular location of nitrate reductase in spinach leaveswas examined by applying an immunocytochemical method. Thinsections were first treated with immunopurified anti-nitratereductase monospecific antibodies, followed by incubation withcolloidal gold-labelled goat anti-rabbit immunoglobulin G asa marker. The nitrate reductase was specifically located inthe chloroplast. When anti-nitrate reductase antibodies wereomitted, or when pre-immune serum was used no label was observed. (Received October 30, 1986; Accepted December 25, 1986)     

The Second Serine Acetyltransferase, Bacterial-type O-Acetylserine (thiol) Lyase and Eukaryotic-type O-Acetylserine (thiol) Lyase from the Primitive Red Alga Cyanidioschyzon merolae

O -acetylserine (thiol) lyase (OASTL) genes were isolated by screening a genomic library of the primitive red alga Cyanidioschyzon merolae and named cmSAT2, and cmOASTL1 and cmOASTL2, respectively. cmSAT2 encoded a polypeptide of 406 amino acids. cmSAT2 was encoded on chromosome IX. cmOASTL1 and cmOASTL2 encoded for polypeptides of 389 and 390 amino acids, respectively. A molecular phylogenic tree of the amino acid sequences suggested that cmOASTL1 belongs to the group of eukaryotic plant OASTL while cmOASTL2 belongs to the bacterial type OASTL. cmOASTL1 and cmOASTL2 were encoded on chromosomes XVII and VIII, respectively. In Northern blot analyses, the probes for cmOASTL1 and cmOASTL2 hybridized with 1.4 kb and 1.3 kb bands, respectively. The identity of cmOASTL1 and cmOASTL2 was confirmed by genetic complementation in an OASTL- deficient mutant of Escherichia coli NK3 using sulfate or sulfide as a sole source of sulfur. Received 19 March 2001/ Accepted in revised form 7 May 2001     

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.Sulfur assimilation in plants is hallmarked by two reaction sequences, namely sulfate reduction and Cys synthesis. The sulfate reduction pathway consists of three steps and produces sulfide from sulfate, which is available in the soil and transported into the roots by specific transporters (Takahashi et al., 2011). Sulfide is subsequently incorporated into the amino acid O-acetylserine (OAS) by O-acetylserine(thiol)lyase (OAS-TL; EC 2.5.1.47) to produce Cys (Hell and Wirtz, 2011). Cys then serves as the sulfur source for all organic metabolites containing reduced sulfur in plants, including proteins, cofactors, and secondary metabolites. The tripeptide glutathione (GSH) is one of the most important Cys-derived metabolites, since it has an important function in redox homeostasis and the control of development (Meyer and Rausch, 2008). Impaired GSH synthesis negatively affects growth of the shoot and root system of Arabidopsis (Arabidopsis thaliana; Vernoux et al., 2000; Xiang et al., 2001), and loss-of-function mutants for the first enzyme (GSH1, Glu-Cys ligase; EC 6.3.2.2) or the second enzyme (GSH2, glutathione synthase; EC 6.3.2.3) of the two-step pathway leading to GSH formation show an embryo- and seedling-lethal phenotype, respectively (Cairns et al., 2006; Pasternak et al., 2008).Cys synthesis by OAS-TL constitutes the direct link between carbon and nitrogen (OAS) as well as sulfur (sulfide) metabolism and, therefore, can be designated as one of the central reactions in plant primary metabolism. The genome of the model plant Arabidopsis encodes nine OAS-TL-like enzymes: OAS-TL A1 (At4g14880), OAS-TL B (At2g43750), and OAS-TL C (At3g59760) are the major isoforms and are localized in the cytosol, plastids, and mitochondria, respectively (Jost et al., 2000). OAS-TL A2 (At3g22460) encodes a truncated and nonfunctional protein (Jost et al., 2000). In the following, therefore, OAS-TL A1 is referred to as OAS-TL A. CYS D1 (At3g04940) and CYS D2 (At5g28020) show OAS-TL activity in vitro (Yamaguchi et al., 2000). Whether they contribute to net Cys synthesis in vivo is unknown (Heeg et al., 2008). CS26 (At3g03630) encodes a plastidic S-sulfocysteine synthase, which prefers thiosulfate instead of sulfide as substrate and produces S-sulfocysteine (Bermúdez et al., 2010). Whether thiosulfate is taken up from the soil or formed within the plant is unclear, but its presence in Arabidopsis was demonstrated (Tsakraklides et al., 2002). However, the synthesis of S-sulfocysteine from thiosulfate potentially constitutes an alternative sulfur fixation pathway. So far, CS26 was shown to be important for the regulation of redox homeostasis in plastids under certain stress conditions (Bermúdez et al., 2010). DES1 (At5g28030; formerly known as CS-LIKE) is a Cys desulfhydrase (EC 4.4.1.15) that releases sulfide in the cytosol (Alvarez et al., 2010). As a Cys-consuming enzyme, it contributes to Cys homeostasis, especially in late vegetative development and under certain stress conditions (Alvarez et al., 2010, 2012). CYS C1 (At3g61440), finally, encodes a mitochondrial β-cyanoalanine synthase (EC 4.4.1.9), which detoxifies cyanide by incorporation into Cys (Yamaguchi et al., 2000; Watanabe et al., 2008a; García et al., 2010). The major isoforms OAS-TL A, OAS-TL B, and OAS-TL C as well as CYS D1 and CYS D2 can interact with serine acetyltransferase (SAT; EC 2.3.1.30) in the cysteine synthase complex (CSC; Heeg et al., 2008). Although SAT acetylates Ser at the hydroxyl group to form OAS, the direct substrate of OAS-TL, formation of the CSC has no substrate-channeling function but contributes to the demand-driven regulation of Cys synthesis (Hell and Wirtz, 2011).The subcellular compartmentation of Cys precursor formation is a remarkable feature of Cys synthesis in higher plants that implies a high degree of regulation between the participating compartments: while sulfate is exclusively reduced to sulfide in plastids (Takahashi et al., 2011), the synthesis of OAS and the incorporation of sulfide take place in all three compartments where SAT and OAS-TL are present, namely in the cytosol, plastids, and mitochondria. Reverse genetics approaches proved a certain redundancy between the different SAT and OAS-TL isoforms, which demonstrates that sulfide, OAS, and Cys can be exchanged between these compartments (Haas et al., 2008; Heeg et al., 2008; Watanabe et al., 2008a, 2008b). Indeed, sulfide can easily diffuse through membranes (Mathai et al., 2009), but OAS and Cys need to be actively transported. However, the identity of these transporters is unknown. Although sulfide, OAS, and Cys can pass the mitochondrial membrane (Wirtz et al., 2012), the loss-of-function mutant for mitochondrial OAS-TL C is the only single oastl knockout mutant that displays a significant growth phenotype (Heeg et al., 2008). This result was astonishing, since OAS-TL C contributes only 5% to extractable foliar OAS-TL activity (Heeg et al., 2008). The retarded growth of the oastlC mutant, however, cannot be explained by the lack of sulfide detoxification in mitochondria by OAS-TL C, due to an alternative detoxification mechanism for sulfide in mitochondria (Birke et al., 2012). These data question the total redundancy between the different OAS-TL isoforms and suggest specific functions in the different subcellular compartments.Despite its central position in the primary metabolism of higher plants, fundamental questions about Cys synthesis are still unanswered. First, the contribution of OAS-TL-like proteins, especially CYS D1, CYS D2, and CS26, to the fixation of sulfur in planta is unknown. Second, the significance of Cys synthesis by the major OAS-TL proteins in the different subcellular compartments during sporophyte and gametophyte development is unclear. In this study, we addressed these questions using a reverse genetics approach. We were able to prove that fixation of sulfur is carried out exclusively by the major OAS-TL isoforms OAS-TL A, OAS-TL B, and OAS-TL C and elucidated specific functions for OAS-TL A in the cytosol and OAS-TL C in mitochondria of leaf cells. Furthermore, we demonstrate that Cys can be supplied by the mother plant for the development of female gametophytes lacking OAS-TL activity. In contrast, the presence of at least one functional OAS-TL isoform is essential in the male gametophyte.     

ATP Sulphurylase and O-Acetylserine Sulphydrylase in Isolated Mesophyll Protoplasts and Bundle Sheath Strands of S-deprived Maize Leaves

In Zea mays L. (cv. XL 72 A) leaves sulphur deficiency causedreduction of soluble protein and chlorophyll contents, whereasATP sulphurylase (EC 2.7.7.4 [EC] ) and O-acetylserine sulphydrylase(EC 4.2.95.9 [EC] ) activities increased with the increasing of S-deprivationtime. The two enzymes exhibited the maximum activity after 5d (ATP sulphurylase) and 3 d (O-acetylserine sulphydrylase)from the beginning of deprivation period. The activities weredifferently distributed between mesophyll protoplasts and bundlesheath strands. The results suggest that the activity of thetwo enzymes may be induced sequentially and differently regulatedin the two types of cells. Key words: ATP sulphurylase, Bundle sheath strands, Mesophyll protoplasts, O-acetylserine sulphydrylase, Sulphur deprivation, Zea     

Sulfate Assimilation in C(4) Plants: Intercellular and Intracellular Location of ATP Sulfurylase, Cysteine Synthase, and Cystathionine beta-Lyase in Maize Leaves

The activity of ATP sulfurylase, cysteine synthase, and cystathionine β-lyase was measured in crude leaf extracts, bundle sheath strands, and mesophyll and bundle sheath chloroplasts to determine the location of sulfate assimilation of C₄ plant leaves. Almost all the ATP sulfurylase activity was located in the bundle sheath chloroplasts while cysteine synthase and cystathionine β-lyase activity was located, in different proportions, in both chloroplast types.

A new spectrophotometric assay for measuring ATP sulfurylase activity is also described.

     

Regulation of the O-acetyl-L-serine(thiol)lyase activity in Monoraphidium braunii

Total level of O-acetyl-L-serine(thiol)lyase (OASTL) activity observed in Monoraphidium braunii fed-repleted cells decreases up to 40% after 24 h the carbon source was removed from the culture; however, no significant change in the activity is observed in N-starved cells. On the other hand, sulfur starvation induces OASTL activity in M. braunii, which may increase 2.5-fold after 36 h. Normal intracellular level of the activity is restored when a sulfur source, such as sulfate, sulfite, L-cysteine, L-methionine or glutathione is added to the culture. The induction of the OASTL activity requires de novo synthesis of protein, and thus the presence in the culture of adequate carbon and nitrogen sources. The OASTL isoenzymes from M. braunii cells are differently affected by S-starvation.     

Mutational analysis of O-acetylserine (thiol) lyase conducted in yeast two-hybrid system

Cysteine biosynthesis, achieved by the sequential reaction of two enzymes, serine acetyltransferase and O-acetylserine (thiol) lyase (OASTL), represents the final step of sulfur assimilation pathway in plants and bacteria. The two enzymes form a bi-enzymatic cysteine synthase complex through specific protein-protein interactions. To identify the amino acids important for cysteine synthase complex formation, several mutations in bacterial OASTL were designed. Effects of mutagenesis were verified in a yeast two-hybrid model that allowed monitoring both, protein-protein interactions and the enzymatic activity of OASTL.     

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.     

Studies on the Growth of Spinach Leaves (Spinacea oleracea)

The growth of spinach leaves has been studied from approximately1 cm long to full size. Over-all growth was measured in termsof area and total number of cells. The differential growth ofleaves was measured by the changes in the shape of squares drawnon the leaf surface. Growth differentials in terms of numbersof cells and number displaying mitotic figures were measuredin leaf discs taken from different positions within leaves. It was found that cell division in spinach leaves continueduntil the leaves reach from one-third to one-half full size.Cell division within the lamina of the leaves was not uniformbut ceased at an early stage of development in the leaf tipregion and continued for an extended period at the base.     

Inhibition of Arabidopsis O-acetylserine(thiol)lyase A1 by tyrosine nitration

The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.     

ATP Sulfurylase Activity in the Soybean [Glycine max (L.) Merr.

ATP sulfurylase activity was assayed in soybean leaf extracts. A simple, rapid assay system using molybdate as an analogue of sulfate was developed. The assay was coupled to inorganic pyrophosphatase. The high pyrophosphatase level in soybean leaf extracts obviated the necessity of adding this enzyme to the assay system. ATP sulfurylase has a pH maximum above 7.5, uses molybdate and ATP as substrates, and requires magnesium ions for activity.     

Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

Key message

This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L.

Abstract

Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a β-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

     

嗜酸氧化亚铁硫杆菌ATP硫酸化酶的表达、纯化及性质鉴定

ATP硫酸化酶是一种催化ATP和SO42-反应生成腺嘌呤-5’-磷酸硫酸(APS)和焦磷酸盐(PPi)的酶,它是硫酸根同化反应第一步的关键酶。以嗜酸氧化亚铁硫杆菌(A.ferrooxidansATCC 23270)基因组为模板,用PCR扩增得到ATPS基因,并克隆到表达载体pLM1上。加入IPTG的诱导表达,用AKTA蛋白纯化仪的镍柱亲和层析纯化得到浓度和纯度都较高的ATPS蛋白。SDS-PAGE分析,证实其分子量大小为33 kD,并成功的测出了其活性,比活达3.0×103U/mg。     

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.