In vitro synthesis of a mutagenic azide metabolite by cell-free bacterial extracts

Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction nor produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by l-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55°C and had a pH optimum between 7–7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Isozyme-Specific Ligands for O-acetylserine sulfhydrylase,a Novel Antibiotic Target

The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.     

Enzymatic synthesis of l-cysteine by O-acetylserine sulfhydrylase of 3-chloro-l-alanine resistant Bacillus sphaericus L-118

The regulatory properties of serine-O-transacetylase and O-acetylserine sulfhydrylase have been investigated with 3-chloro-l-alanine resistant Bacillus sphaericus L-118. The enhancement of O-acetylserine sulfhydrylase formation by 3-chloro-l-alanine was observed and this effect was counteracted by corepressor l-cysteine. O-Acetylserine sulfhydrylase occurring in B. sphaericus L-118 can catalyse β-replacement reaction of 3-chloro-l-alanine in the presence of a high concentration of sodium hydrosulfide to form l-cysteine. The optimal reaction conditions for l-cysteine production were studied using resting cells. Under optimal conditions, about 80% of the added 3-chloro-l-alanine could be converted to l-cysteine. The highest yield achieved was 70 mg of l-cysteine per 1.0 ml of the reaction mixture.     

Role of o-acetylserine in hydrogen sulfide emission from pumpkin leaves in response to sulfate

In the presence of excess sulfate, cysteine synthesis in pumpkin (Cucurbita pepo) leaves is not limited by sulfate reduction, but by the availability of O-acetylserine. Feeding of O-acetylserine or its metabolic precursors S-acetyl-coenzyme-A and coenzyme A to leaf discs enhanced the incorportion of [³⁵S]sulfate into reduced sulfur compounds, mainly into cysteine, at the cost of lowered H₂S emission; the uptake and reduction of sulfate is not affected by these treatments. β-Fluoropyruvate, an inhibitor of the generation of S-acetyl-coenzyme A via pyruvate dehydrogenase, stimulated H₂S emission in response to sulfate. This stimulation is overcompensated by addition of O-acetylserine, S-acetyl-coenzyme A, or coenzyme A. These results indicate that, in the presence of high amounts of sulfate, excess sulfur is reduced and emitted as H₂S into the atmosphere. The H₂S emitted seems to be produced by liberation from a precursor of cysteine rather than by cysteine desulfhydration.     

Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized.     

Purification of serine acetyltransferase,a component of a multienzyme complex,by immunoadsorption and selective dissociation of the complex

An immunoadsorbent column chromatography procedure utilizing antibody to one component of a multienzyme complex has been utilized for purification of the second component of this complex. Rabbit immunoglobulin G (IgG) specific for the O-acetylserine sulfhydrylase component of the multienzyme complex cysteine synthetase was linked to Sepharose 4B resin. A crude preparation of cysteine synthetase was bound to a column of IgG-Sepharose, other proteins were removed by washing, and the serine acetyltransferase component of the complex was eluted with 50 mmO-acetylserine, which dissociates the complex of the two enzymes. This purification step produces about a 400-fold increase in specific activity.     

salmonella typhimurium/enzymol.

Cysteine synthetase from Salmonella typhimurium LT-2 displays a saturation curve for sulfide identical to that obtained with uncomplexed O-acetylserine sulfhydrylase, indicating substrate inhibition with a K_m of 0.1 ± 0.017 mm and a K₁ of 0.303 ± 0.194 mm. With both l-serine and acetyl CoA, however, cysteine synthetase exhibits two intermediary plateaus in the respective saturation curves. The time course of cysteine synthetase activity when the reaction is started by adding enzyme displays a pronounced lag phase. This lag is explained as being due to the buildup of a sufficient concentration of O-acetyl-l-serine to permit binding to O-acetylserine sulfhydrylase. This conclusion is substantiated by the fact that plots of $\text{1}\text{τ}$ against concentrations of both l-serine and acetyl CoA reflect the saturation curves for these substrates. In addition, the incubation of the complex with l-serine and acetyl CoA results in the accumulation of the intermediate products of the reaction sequence, CoA and O-acetyl-l-serine. Dissociation of the multienzyme complex under these conditions was ruled out by Sephadex G-200 chromatography of the complex after incubation with assay levels of the substrates of the reaction. Aggregation of cysteine synthetase was detected using disc gel electrophoresis and confirms earlier reports [Kredich, N. M., and Tomkins, G. M. (1966) J. Biol. Chem.241, 4955–4965]. Aggregation of O-acetylserine sulfhydrylase was also detected using the same technique.     

Regulation of Sulfate Assimilation in Tobacco Cells: EFFECT OF NITROGEN AND SULFUR NUTRITION ON SULFATE PERMEASE AND O-ACETYLSERINE SULFHYDRYLASE

The effect of nitrogen and sulfur nutrition on sulfate permease and O-acetylserine sulfhydrylase was studied in tobacco cells.     

Serine O-acetyltransferase is important,but not essential for cysteine–methionine synthesis in Fusarium graminearum

O-acetyltransferase (SAT) is a key enzyme converting serine into O-acetylserine in the synthesis of sulphur-containing amino acids. To characterize the function of FgSAT in Fusarium graminearum, three deletion mutants of FgSAT (ΔFgSAT-1, -2 and -18) were obtained using a gene replacement strategy. The three mutants did not show recognizable phenotypic changes on potato dextrose agar medium, but exhibited a very weak growth on fructose gelatin agar (FGA) medium containing SO₄ ^2? as sole sulfur source. Supplementation of O-acetylserine, cysteine, or methionine, but not serine, rescued the defect of mycelial growth in FgSAT deletion mutants, indicating that FgSAT is involved in conversion of serine into O-acetylserine. The three mutants had a decrease in conidiation in mung bean liquid, but not in carboxymethyl cellulose. Virulence, deoxynivalenol production and fungicide sensitivity assays found that the three mutants showed no significant difference from wild-type progenitor PH-1. Real-time PCR assays detected an increase in expression levels of FgOAHS, FgCBS and FgCGL genes involved in the alternative pathway in FgSAT deletion mutants, suggesting that the alternative pathway in F. graminearum is present and can operate. Addition of homoserine, the upstream substrate of the alternative pathway, also restored the normal mycelial growth of FgSAT deletion mutants on FGA, indicating that the alternative pathway in F. graminearum might be positively regulated by homoserine.     

Oxidation versus Reductive Detoxification of SO(2) by Chloroplasts

Intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) both oxidized and reduced added sulfite in the light. Oxidation was fast only when endogenous superoxide dismutase was inhibited by cyanide. It was largely suppressed by scavengers of oxygen radicals. After addition of O-acetylserine, chloroplasts reduced sulfite to cysteine and exhibited sulfite-dependent oxygen evolution. Cysteine synthesis from sulfite was faster than from sulfate. The results are discussed in relation to species-specific differences in the phytotoxicity of SO₂.     

Assessing functional diversity in the soybean β-substituted alanine synthase enzyme family

In plants, proteins of the β-substituted alanine synthase (BSAS) enzyme family perform a diverse range of reactions, including formation of cysteine from O-acetylserine and sulfide, detoxification of cyanide by its addition to cysteine, the breakdown of cysteine into pyruvate, ammonia, and sulfide, and the synthesis of S-sulfocysteine. With the completed genome sequence of soybean (Glycine max (L.) Merr. cv. Williams 82), the functional diversity of the BSAS in this highly duplicated plant species was examined to determine whether soybean BSAS enzymes catalyze the various reactions connected to cysteine metabolism. The 16 soybean BSAS can be grouped into clades that are similar to those observed in Arabidopsis. Biochemical analysis of soybean BSAS proteins demonstrate that enzymes of clades I and III function as O-acetylserine sulfhydrylases for cysteine synthesis, clade II encodes cysteine desulfhydrase activity, and that clade V proteins function as β-cyanoalanine synthase for cyanide detoxification. Although clade IV is similar to Arabidopsis S-sulfocysteine synthase, this activity was not detected in the soybean homolog. Overall, our results show that bioinformatics approach provides a useful method to assess the biochemical properties of BSAS enzymes in plant species.     

The synthesis of a mannosyl-N-acetylglucosamine-l-asparagine compound: 2-acetamido-N-(l-aspart-4-oyl)-2-deoxy-3-O-α-d-mannopyranosyl-β-d-glucopyranosylamine

The title compound, used in the synthesis of glycopeptides and as a reference substance in the structural elucidation of glycoproteins, was synthesized by condensation of 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide with 2-acetamido-4,6-O-benzylidene-α-d-glucopyranosyl azide, followed by removal of the benzylidene group to give the disaccharide azide 6 and acetylation. The resulting fully acetylated disaccharide azide 7 was also obtained by treatment of the known 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-glucopyranose with hydrogen chloride and then with silver azide. The azide 7 was reduced in presence of platinum oxide (Adams' catalyst), and the resulting amine was condensed with 1-benzyl N-benzyloxycarbonyl-l-aspartate in the presence of N,N′-dicyclocarbodiimide. The removal of the protective group was accomplished by hydrogenolysis and O-deacetylation. In a second route, the disaccharide azide 6 was reduced and then condensed with 1-benzyl N-benzyloxycarbonyl-l-aspartate, and the resulting product hydrogenolyzed.     

The synthesis of oligosaccharide-asparagine compounds. V. O- -D-mannopyranosyl-(1 leads to 6)-O-(2-acetamido-2-deoxy- -D-glucopyranosyl)-(1 leads to 4)-2-acetamido-N-(L-aspart-4-oyl)-2-deoxy- -D-glucopyranosylamine

O-α-d-Mannopyranosyl-(1→6)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→4)-2-acetamido-N-(l-aspart-4-oyl)-2-deoxy-β-d-glucopyranosylamine (12), used in the synthesis of glycopeptides and as a reference compound in the structure elucidation of glycoproteins, was synthesized via condensation of 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide with 2-acetamido-4-O-(2-acetamido-3-O-acetyl-2-deoxy-β-d-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-β-d-glucopyranosyl azide (5) to give the intermediate, trisaccharide azide 7. [Compound 5 was obtained from the known 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-β-d-glucopyranosyl azide by de-O-acetylation, condensation with benzaldehyde, acetylation, and removal of the benzylidene group.] The trisaccharide azide 6 was then acetylated, and the acetate reduced in the presence of Adams' catalyst. The resulting amine was condensed with 1-benzyl N-(benzyloxycarbonyl)-l-aspartate, and the O-acetyl, N-(benzyloxycarbonyl), and benzyl protective groups were removed, to give the title compound.     

Oxidation and reduction of sulfite by chloroplasts and formation of sulfite addition compounds

After exposing intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) and capable of photoreducing CO₂ at high rates to different concentrations of radioactive sulfite in the light or in the dark, ³⁵SO₂ and H₂³⁵S were removed from the acidified suspensions in a stream of nitrogen. Remaining activity could be fractionated into sulfate, organic sulfides, and sulfite addition compounds. When chloroplast suspensions contained catalase, superoxide dismutase and O-acetylserine, the oxidation of sulfite to sulfate was slower in the light than the reductive formation of sulfides that exhibited a maximum rate of about 2 micromoles per milligram chlorophyll per hour, equivalent to about 1% of maximum carbon assimilation. Botht the oxidative and the reductive detoxification of sulfite were very slow in the dark. Oxidation was somewhat, but not much, accelerated in the light in the absence of O-acetylserine, which caused a dramatic decrease in the formation of organic sulfides and an equally dramatic increase in the concentration of sulfite addition compounds whose formation was light-dependent. The sulfite addition compounds were not identified. Addition compounds did not accumulate in the dark. In the light, the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, decreased not only the reduction, but also the oxidation of sulfite and the formation of addition compounds.     

Effect of sodium azide on sister-chromatid exchanges in human lymphocytes and Chinese hamster cells

Previous reports from this laboratory and others indicate that sodium azide is a unique mutagen. It is highly mutagenic in S. typhimurium TA1530 as well as in barley, rice, peas, yeast and Chinese hamster V79 cells. However, azide apparently does not produce chromosome breaks in barley, Vicia or human lymphocytes. Therefore, a study of the effects of azide on sister-chromatid exchanges (SCE) appeared warranted.Human whole blood and Chinese hamster K1 cell line were exposed for 4 and 2 h resp. to various concentrations of sodium azide ranging from 10⁻³ to 10⁻⁷ M. Cells were harvested and chromosomes stained by the FPG technique. In human lumphocytes, concentrations above 10⁻⁴ induced lethality whereas the K1 cell line was sensitive to concentrations above 10⁻⁵ M. The lower concentrations of azide produced no significant increase in SCE frequency above controls. Concurrent mitomycin C treatments produced significant increases in SCE levels.This apparent lack of induction of SCEs above background combined with previous data demonstrating negative clastogenic but very positive mutagenic activity of azide confirms the uniqueness of this mutagen. It would appear that azide is one of the few known potent mutagens that does not increase SCEs and/or break chromosomes.     

Mutagenicity of nitrofuran derivatives, including furylfuramide, a food preservative.

The effects of sodium azide (NaN₃) in combination with diethyl sulfate (dES) or N-methyl-N′-nitrosourea (MNH) on mutation frequency in barley were studied. It was found that sodium azide produced high frequencies of chlorophyll mutations when used alone and has a synergistic effect on mutation yields following MNH treatments. However, the mutation frequency was decreased whe azide was applied following dES treatment of seeds. The mutagenic efficiency of azide was found to be high, possibly because of low “physiological” damage. The synergistic increase in mutation yields by MNH and azide treatment indicates that azide has unusual promise as a mutagen for both practical and research applications.     

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