Analysis of some enzymes activities of hydrogen sulfide metabolism in plants.

Hydrogen sulfide (H2S) which is considered as a novel gasotransmitter after reactive oxygen species and nitric oxide in plants has dual character, that is, toxicity that inhibits cytochrome oxidase at high concentration and as signal molecule which is involved in plant growth, development, and the acquisition of tolerance to adverse environments such as extreme temperature, drought, salt, and heavy metal stress at low concentration. Therefore, H2S homeostasis is very important in plant cells. The level of H2S in plant cells is regulated by its synthetic and degradative enzymes, L-/D-cysteine desulfhydrase (L-/D-DES), sulfite reductase (SiR), and cyanoalanine synthase (CAS), which are responsible for H2S synthesis, while cysteine synthase (CS) takes charge of the degradation of H2S, but its reverse reaction also can produce H2S. Here, after crude enzyme is extracted from plant tissues, the activities of L-/D-DES, SiR, CAS, and CS are measured by spectrophotometry, the aim is to further understand homeostasis of H2S in plant cells and its potential mechanisms.

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 ß-substituted alanine synthases.

Enzymes of cysteine synthesis show extensive and conserved modifications patterns that include N(α)-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.

K30, H150, and H168 are essential residues for coordinating pyridoxal 5'-phosphate of O-acetylserine sulfhydrylase from Acidithiobacillus ferrooxidans.

O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV-vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5'-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization.

Crystallization and preliminary crystallographic analysis of cysteine synthase from Entamoeba histolytica.

Entamoeba histolytica, the causative agent of human amoebiasis, is essentially anaerobic, requiring a small amount of oxygen for growth. It cannot tolerate the higher concentration of oxygen present in human tissues or blood. However, during tissue invasion it is exposed to a higher level of oxygen, leading to oxygen stress. Cysteine, which is a vital thiol in E. histolytica, plays an essential role in its oxygen-defence mechanisms. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine-biosynthetic pathway, which involves cysteine synthase (EhCS) as a key enzyme. In this study, EhCS was cloned, expressed in Escherichia coli and purified by affinity and size-exclusion chromatography. The purified protein was crystallized in space group P4(1) with two molecules per asymmetric unit and a complete data set was collected to a resolution of 1.86 A. A molecular-replacement solution was obtained using the Salmonella typhimurium O-acetylserine sulfhydrylase structure as a probe and had a correlation coefficient of 37.7% and an R factor of 48.8%.

Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli.

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.

Structure of the O-acetylserine sulfhydrylase isoenzyme CysM from Escherichia coli.

The enzyme O-acetylserine sulfhydrylase participates in the biosynthesis of l-cysteine in bacteria and plants. The structure of isoenzyme B (CysM) from Escherichia coli was established in a hexagonal crystal form at 2.7 A resolution (wild-type) and in a merohedrally twinned tetragonal crystal form at 2.1 A resolution (surface mutant). Structural superpositions revealed the variations with respect to isoenzyme A (CysK) and explained the different substrate specificities. A geometric model of the reaction catalyzed by CysM is proposed. Both isoenzymes are used for the production of l-amino acid derivatives as building blocks for the synthesis of peptides and peptidomimetic drugs. Since the structure of CysM revealed a remarkable main chain variation at the active center, it constitutes a further starting point for engineering mutants with novel substrate specificities.

Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V.

The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.

Purification and characterization of O-Acetylserine sulfhydrylase of Corynebacterium glutamicum.

We highly purified O-acetylserine sulfhydrylase from the glutamate-producing bacterium Corynebacterium glutamicum. The molecular mass of the purified enzyme was 34,500 as determined by SDS-polyacrylamide gel electrophoresis, and 70,800 as determined by gel filtration chromatography. It had an apparent Km of 7.0 mM for O-acetylserine and a Vmax of 435 micromol min-1 (mg x protein)-1. This is the first report of the cysteine biosynthetic enzyme of C. glutamicum in purified form.

Anti-stress activity of Propionibacterium freudenreichii : identification of a reactivative protein.

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.

Cysteine synthase of an extremely thermophilic bacterium, Thermus thermophilus HB8.

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.

Beta-cyanoalanine synthase and cysteine synthase from potato: molecular cloning, biochemical characterization, and spatial and hormonal regulation.

Beta-cyanoalanine synthase (CAS, L-3-cyanoalanine synthase; EC 4.4.1.9) is the most important enzyme in cyanide metabolism. In addition to CAS, cysteine synthase (CS, EC 4.2.99.8) possesses CAS activity. To explore the physiological significance of cyanide metabolism, we isolated the cDNA clones corresponding to purified CAS (designated PCAS-1 and PCAS-2) and CS (designated PCS-1 and PCS-2) from potato using the information of these amino acid sequences. The recombinant proteins of PCS-1, PCS-2 and PCAS-1 catalyzed both CAS and CS reactions, although the ratios between CAS and CS activity were remarkably different. PCAS-1 preferred the substrates for the CAS reaction to the substrates for the CS reaction. From the kinetic characters and homology of amino acid sequences with known CS-like proteins, PCS-1, PCS-2 and PCAS-1 were identified as cytosolic CS, plastidic CS and mitochondrial CAS, respectively. The highest level of CAS activity, CAS protein and its mRNA were detected in potato buds. Stimulation of CAS activity and protein accumulation by ethylene without the concomitant increase of its mRNA suggested that ethylene induces CAS protein accumulation at the post-transcriptional level.

Cysteine synthase (O-acetylserine (thiol) lyase) substrate specificities classify the mitochondrial isoform as a cyanoalanine synthase.

A cyanoalanine synthase and two isoforms (A, cytosolic and B, chloroplastic) of cysteine synthase (O:-acetylserine (thiol) lyase) were isolated from spinach. N-terminal amino acid sequence analysis of the cyanoalanine synthase gave 100% homology for the determined 12 residues with a published sequence for the mitochondrial cysteine synthase isoform. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions, although with different efficiencies. Michaelis-Menten kinetics were observed for all three enzymes when substrate saturation experiments were performed varying O:-acetylserine, chloroalanine and cysteine. Negative co-operative kinetics were observed for cysteine synthases A and B when substrate saturation experiments were performed varying sulphide and cyanide, compared with the Michaelis-Menten kinetics observed for cyanoalanine synthase. The exception was negative co-operativity observed towards sulphide for cyanoalanine synthase with O:-acetylserine as co-substrate. The optimum sulphide concentration was dependent on the alanyl co-substrate used. The amino acid sequence similarity places these three enzymes in the same gene family, and whilst the close kinetic similarities support this, they also indicate distinct roles for the isoforms.

Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase.

Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively. Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A. thaliana genome. Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E. coli clearly showed cysteine synthase activity. In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not. Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo. The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A. thaliana seedlings were subjected to various stresses, including nutrient deprivation. In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme.

Purification and characterization of beta-cyanoalanine synthase and cysteine synthases from potato tubers: are beta-cyanoalanine synthase and mitochondrial cysteine synthase same enzyme?

beta-Cyanoalanine synthase (CAS; EC 4.4.1.9) and two kinds of cysteine synthases (CS; EC 4.2.99.8) have been purified from the particulate fraction of potato tubers. By DEAE Sephacel and Resource PHE chromatography, CAS activity was separated from two CS activities, designated as CS-1 and CS-2. The molecular masses of CAS, CS-1 and CS-2 were estimated to be 37, 39 and 34 kDa, respectively, by SDS-PAGE analysis. The purified CAS had CS activity, and both CS-1 and CS-2 had CAS activity. However, CAS and CSs had significant differences in kinetic characters. The antibody raised against purified CAS discriminated CAS from CSs, whereas the antibody raised against purified CS-2 recognized CS-1 and CS-2 but not CAS. The molecular mass and the partial amino acid sequence of CS-2 were similar to those of the cytosolic CS of potato, whereas the molecular mass of CS-1 was similar to that of the plastidic CS. The partial amino acid sequence of CAS was similar to those of CS isozymes, especially the mitochondrial CS isolated from spinach.

Characterization of O-acetyl-L-serine sulfhydrylase purified from an alkaliphilic bacterium.

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) activity was shown to be very high compared with O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity and L-cystathionine cleaving activities, in an extract of cells of an alkaliphilic bacterium grown in a synthetic medium. The synthesis of the first enzyme was repressed by approximately 55% by both L-cystine and L-djenkolic acid added to the medium at a concentration of 0.5 mM, but L-methionine (1 mM) and S-adenosyl-L-methionine (0.5 mM) affected it to lesser extents. Its enzyme activity was inhibited by 25% and 12% by methionine (10 mM) and S-adenosylmethionine (5 mM), respectively. The enzyme was purified from the extract through ammonium sulfate fractionation, heat treatment, and chromatography on columns of DEAE-cellulose, Sephacryl S-300, and Octyl Sepharose CL-4B with a recovery of 21%. Polyacrylamide gel electrophoresis with sodium dodecylsulfate of the preparation obtained finally showed its homogeneity and the molecular mass of 37,000 Da for dissociated subunits. Gel filtration of the enzyme on a Sephacryl S-300 column showed an approximate molecular mass of 72,000 Da, suggesting that the enzyme was comprised of two identical subunits. The enzyme catalyzed the beta-replacement reaction with O-acetylserine as a substrate, and showed no reactivity to other O-substituted amino acids tested. The reaction proceeded best at 40 degrees C (when tested at pH 7.5), and at pH 6.5 (at 40 degrees C). The enzyme kept 90% its activity after incubation at 65 degrees C (at pH 7.5) for 30 min, and more than 90% after 30 min incubation at pHs 7-12 at 30 degrees C. The enzyme had a Km of 4 mM for O-acetyl-L-serine and a Vmax of 37.0 micromol/min/mg of protein, a very low value compared with those of other organisms. However, the content of the enzyme in the extract was calculated to be approximately 3.5% total protein. Sensitivity of the enzyme to carbonyl reagents was very low, although it was shown to have pyridoxal 5'-phosphate as a cofactor by examination of its absorption spectrum. Sulfhydryl reagents tested showed no inhibition. The novelty of this enzyme among analogous sulfhydrylases purified from other organisms was discussed.

Purification and characterization of serine acetyltransferase from Escherichia coli partially truncated at the C-terminal region.

Incubation of serine acetyltransferase (SAT) from Escherichia coli at 25 degrees C in the absence of protease inhibitors yielded a truncated SAT. The truncated SAT was much less sensitive to feedback inhibition than the wild-type SAT. Analyses of the N- and C-terminal amino acid sequences found that the truncated SAT designated as SAT delta C20 was a resultant form of the wild-type SAT cleaved between Ser 253 and Met 254, deleting 20 amino acid residues from the C-terminus. Based on these findings, we constructed a plasmid containing an altered cysE gene encoding the truncated SAT. SAT delta C20 was produced using the cells of E. coli JM70 transformed with the plasmid and purified to be homogeneous on an SDS-polyacrylamide gel. Properties of the purified SAT delta C20 were investigated in comparison with those of the wild-type SAT and Met-256-Ile mutant SAT, which was isolated by Denk and Böck but not purified (J. Gen. Microbiol., 133, 515-525 (1987)). SAT delta C20 was composed of four identical subunits like the wild-type SAT and Met-256-Ile mutant SAT. Specific activity, optimum pH for reaction, thermal stability, and stability to reagents for SAT delta C20 were similar those for the wild-type SAT and Met-256-Ile mutant SAT. However, SAT delta C20 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A), a counterpart of the cysteine synthetase and did not reduce OASS activity in contrast to the wild-type SAT and Met-256-Ile mutant SAT.

Purification and characterization of a novel cysteine synthase isozyme from spinach hydrated seeds.

A novel type of cysteine synthase (CSase, EC 4.2.99.8) isozyme, designated as CSase 1', was purified to homogeneity from hydrated spinach seeds. The enzyme had a molecular weight of 68,000 and consisted of two identical subunits of M(r), 34,000. The apparent K(m) for O-acetyl-L-serine was 8.33 mM and that for sulfide was 0.66 mM. The activity of CSase 1' was maintained when it was treated at 60 degrees C for 1 min. This novel enzyme was similar to CSases 1, 2, and 3 already purified from spinach leaves, in results of double immunodiffusion, molecular weight, subunit composition, K(m) values for O-acetyl-L-serine and sulfide, and heat stability. On the other hand, N-terminal amino acid sequence, effects of immunotitration, pH optimum, and effects of hydroxylamine on purified CSase 1' were different from those of the other CSases. Furthermore, it was found that CSases 2S and 3S isolated from hydrated spinach seeds were identical with the CSases 2 and 3 reported previously. It was also disclosed that CSases 1, 2, and 3 were localized in chloroplasts, cytosol, and mitochondria, respectively.

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.