Sulfate Assimilation Pathway Research Articles

Mutations affecting the sulphur assimilation pathway in Aspergillus nidulans: their effect on sulphur amino acid metabolism.

Several sul-reg mutants of Aspergillus nidulans isolated as constitutive for arylsulphatase were studied with respect to the regulation of enzymes involved in cysteine and homocysteine synthesis and to the pool of sulphur amino acids. All mutants examined showed a decreased concentration of glutathione as compared with the wild type, and all mutants, with one exception, had a decreased total pool of sulphur amino acids. The results suggest that the mutants are leaky in the sulphate assimilation pathway. They show derepression of cysteine synthase, homocysteine synthase, cystathionine beta-synthase and gamma-cystathionase. In spite of having derepressed homocysteine synthase, the enzyme which constitutes an alternative pathway for homocysteine synthesis, the sul-reg mutations do not suppress lesions in genes required for the main homocysteine-synthesizing pathway. This indicates that the derepression of homocysteine synthase is not in itself sufficient for physiological functioning of this enzyme, but seems to depend also on the effectiveness of cysteine synthesis and sulphide formation.

Occurrence and evolutionary significance of two sulfate assimilation pathways in the rhodospirillaceae

The ability to use adenosine 5′-phosphosulfate (APS) or 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the substrate for the initial reductive step in sulfate assimilation has been tested in most of the known Rhodospirillaceae species and in some chemotrophic bacteria. Improved and optimized methods for the synthesis and purification of the sulfonucleotides APS and PAPS are described. The production of acid volatile radioactivity from 35S-APS and 35S-PAPS was measured under various conditions in the presence and absence of non-labeled sulfate. Specific differences in the ability to reduce APS or PAPS were observed among the Rhodospirillaceae species and also the chemotrophic bacteria. APS was found to be the substrate of the thiolsulfotransferase in Rps. acidophila, Rps. globiformis, Rm. vannielii, Rc. purpureus, R. tenue, Rps. gelatinosa, in Alcaligenes eutrophus and Pseudomonas aeruginosa. PAPS was the substrate in Rps. capsulata, Rps. sphaeroides, Rps. sulfidophila, Rps. palustris, Rps. viridis, R. rubrum, R. fulvum, in Paracoccus denitrificans and in several Enterobacteriaceae. The presence of different enzymatic systems for sulfate reduction in the Rhodospirillaceae family is compared with their taxonomical grouping and their possible phylogenetic relatedness.

Emission of Hydrogen Sulfide from Sulfur Dioxide-Fumigated Pine Trees

Pine (Pinus silvestris L.) trees subjected to relatively low concentration of SO(2) in the field emit H(2)S from the needles, as demonstrated by gas chromatographic analysis after preconcentration on a molecular sieve. H(2)S is the only reduced sulfurous compound emitted from SO(2) fumigated leaves. The emission is light and SO(2) concentration dependent. Pine trees in the field and in laboratory experiments continue to emit H(2)S several hours after the termination of prolonged SO(2) fumigation. The maximum emission rates observed from pine trees in the field and in laboratory experiments, 14 and 20 nanomoles per milligram chlorophyll per hour respectively, are about the activity expected for the sulfur assimilation pathway in the chloroplasts.

Evidence for an intracellular sulfur cycle in cucumber leaves

H2S emission from cucumber (Cucumis sativus L.) leaf discs supplied with L-cysteine in the dark is inhibited 80-90% by aminooxyacetic acid (AOA), an inhibitor of pyridoxal-phosphate dependent enzymes. Exposure to L-cysteine in the light enhanced the emission of H2S in response to this sulfur source. Turning off the light reduced the emission of H2S to the rate observed in continuous dark; turning on the light enhanced the emission of H2S to the rate observed in continuous light. Therefore, in the light H2S emission in response to L-cysteine becomes a partially light-dependent process. Treatment with cyanazine, an inhibitor of photosynthetic electron transport, reduced H2S emission in the light to the rate observed in continuous dark, but did not affect H2S emission in the dark. In leaf discs pre-exposed to L-cysteine in the light, treatment with cyanazine+ AOA inhibited the emission of H2S in response to L-cysteine completely. Therefore, only part of the H2S emitted in response to this sulfur source is derived from a light-independent, but pyridoxal-phosphate-dependent process; the balance of the H2S emitted is derived from a light-dependent process that can be inhibited by cyanazine. When cucumber leaf discs were supplied with a pulse of L-[(35S)]cysteine, radioactively labeled H2S was emitted in two waves, one during the first hour of exposure to L-cysteine, and a second after 3-4 h; unlabeled H2S, however, was emitted continuously. The second wave of emission of labeled H2S was not observed in pulse-chase experiments in which sulfate or cyanazine were added to the treatment solution after 3 h of exposure to L-cysteine, or when the lights was turned off. The labeling pattern of sulfur compounds inside cucumber cells supplied with a pulse of L-[(35)S]cysteine showed that the labeled H2S released from L-cysteine partially enters first the sulfite, then the sulfate pool of the cells. The radioactively labeled sulfate, however, is not incorporated into L-cysteine, but enters the H2S pool of the cells again. These observations are consistent with the idea of an intracellular sulfur cycle in plant cells. The L-cysteine taken up by the leaf discs seems to be desulfhydrated in a light-independent, but pyridoxal-phosphate-dependent process. The H2S synthesized this way may be partially released into the atmosphere; the other part of the H2S produced in response to L-cysteine may be oxidized to sulfite, then to sulfate, which is subsequently reduced via the light-depent sulfate assimilation pathway. In the presence of excess L-cysteine, synthesis of additional cysteine may be inhibited, and the sulfide moiety may be split off carrier bound sulfide to enter the H2S pool of the cells again. It is suggested that the function of this sulfur cycle may be regulation of the free cysteine pool.

Sulfate assimilation in Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to grow on sulfate as sole source of sulfur, but only at concentrations below 1 mM. Good growth was observed with thiosulfate, cysteine or methionine as sulfur sources. Tetrathionate supported slow growth. Sulfide and sulfite were growth inhibitory. Growth inhibition by higher sulfate concentrations was overcome by the addition of O-acetylserine, which is known as derepressor of sulfate-assimilating enzymes, and by reduced glutathione. All enzymes of the sulfate assimilation pathway. ATP-sulfurylase, adenylylphosphate-sulfotransferase, thiosulfonate reductase and O-acetylserine sulfhydrylase are present in R. globiformis. Sulfate was taken up by the cells and the sulfur incorporated into the amino acids cysteine, methionine and homocysteine. It is concluded, that the failure of R. globiformis to grow on higher concentrations of sulfate is caused by disregulation of the sulfate assimilation pathway. Some preliminary evidence for this view is given in comparing the activities of some of the involved enzymes after growth on different sulfur sources and by examining the effect of O-acetylserine on these activities.

Regulatory coupling of nitrate and sulfate assimilation pathways in cultured tobacco cells

When cultured tobacco cells are provided growth-limiting concentrations of sulfur as sulfate, the rate of development of nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) is proportional to the initial sulfate concentration. When the cells are provided growth-limiting concentrations of nitrogen as nitrate, the rate of derepression of ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) is proportional to the initial nitrate concentration. These results are taken to be indicative of a reciprocal regulatory coupling between the nitrate and sulfate assimilation pathways.

Light-dependent Emission of Hydrogen Sulfide from Plants

With the aid of a sulfur-specific flame photometric detector, an emission of volatile sulfur was detected from leaves of cucumber (Cucumis sativus L.), squash and pumpkin (Cucurbita pepo L.), cantaloupe (Cucumis melo L.), corn (Zea mays L.), soybean (Glycine max [L.] Merr.) and cotton (Gossypium hirsutum L.). The emission was studied in detail in squash and pumpkin. It occurred following treatment of the roots of plants with sulfate and was markedly higher from either detached leaves treated via the cut petiole, or whole plants treated via mechanically injured roots. Bisulfite elicited higher rates of emission than sulfate. The emission was completely light-dependent and increased with light intensity. The rate of emission rose to a maximum and then declined steadily toward zero in the course of a few hours. However, emission resumed after reinjury of roots, an increase in light intensity, an increase in sulfur anion concentration, or a dark period of several hours.The emission was identified as H(2)S by the following criteria: it had the odor of H(2)S; it was not trapped by distilled H(2)O, but was trapped by acidic CdCl(2) resulting in the formation of a yellow precipitate, CdS; it was also trapped by base and the contents of the trap formed methylene blue when reacted with N,N-dimethyl-p-phenylenediamine and Fe(3+).H(2)S emission is not the cause of leaf injury by SO(2), since bisulfite produced SO(2) injury symptoms in dim light when H(2)S emission was low, while sulfate did not produce injury symptoms in bright light when H(2)S emission was high.The maximum rates of emission observed, about 8 nmol min(-1) g fresh weight(-1), are about the activity that would be expected for the sulfur assimilation pathway of a normal leaf. H(2)S emission may be a means by which the plant can rid itself of excess inorganic sulfur when HS(-) acceptors are not available in sufficient quantity.

Sulfate Uptake in Saccharomyces cerevisiae : Biochemical and Genetic Study

Sulfate uptake is the first step of the sulfate assimilation pathway, which has been shown in our laboratory to be part of the methionine biosynthetic pathway. Kinetic study of sulfate uptake has shown a biphasic curve in a Lineweaver-Burk plot. The analysis of this plot indicates that two enzymes participate in sulfate uptake. One (permease I) has a high affinity for the substrate (K(m) = 0.005 mM); the other (permease II) shows a much lower affinity for sulfate (K(m) = 0.35 mM). Regulation of the synthesis of both permeases is under the control of exogenous methionine or S-adenosylmethionine. It was shown, moreover, that synthesis of sulfate permeases is coordinated with the synthesis of the other methionine biosynthetic enzymes thus far studied in our laboratory. An additional specific regulation of sulfate permeases by inhibition of their activity by endogenous sulfate and adenosyl phosphosulfate (an intermediate metabolite in sulfate assimilation) has been shown. A mutant unable to concentrate sulfate has been selected. This strain carried mutations in two independent genes. These two mutations, separated in two different strains, lead to modified kinetics of sulfate uptake. The study of these strains leads us to postulate that there is an interaction in situ between the products of these two genes.

Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. Effects of sulfur and nitrogen sources on the formation and decay of the enzyme.

The ATP sulfurylase of cultured tobacco cells is repressed during growth on readily assimiliated sulfur sources, such as sulfate, L-cysteine, or L-methionine, but it is derepressed during growth on slowly assimiliated sulfur sources, such as L-djenkolate or glutathione, or during sulfur starvation. The enzyme is not induced by sulfate. The enzyme level in the cells begins to rise 12 to 24 h after the derepression conditions are initiated and continues to rise for 3 to 4 days, up to as much as 25 times above the initial specific activity. Addition of a repressing sulfur source to derepressed cells causes the enzyme to decay. Derepression by sulfur limitation does not occur in cells starved for nitrogen, a circumstance in which turnover synthesis of protein is known to continue. Upon addition of a nitrogen source to such cells, the development of the enzyme begins within 12 h, along with the resumption of growth and net protein synthesis. Derepression occurs in cells growing on the slowly assimilated nitrogen in urea, reaching specific activities very similar to those which develop in cells grown on nitrate, in spite of the lower protein accumulation rate on urea. Thus the ATP sulfurylase of tobacco cells appears to be regulated by both a negative feedback mechanism in which an end product of the sulfate assimilation pathway is the effector, and by a positive mechanism which serves to couple the regulation of the sulfate assimilation pathway to the cells' potential for nitrogen assimilation, i.e. net protein synthesis. The sulfur compounds which affect the development of ATP sulfurylase in vivo have no effect on the enzyme activity in vitro. Furthermore, extracts with high activity contain no activator and extracts with low activity contain no inhibitor of ATP sulfurylase. Cycloheximide, at a concentration which strongly inhibits amino acid incorporation into protein, inhibits derepression. ATP sulfurylase does not decay in cells inhibited by cycloheximide. Therefore, the changes in ATP sulfurylase of tobacco cells appear to involve changes in the rate of formation or degradation of the enzyme.

Derepression of ATP sulfurylase by the sulfate analogs molybdate and selenate in cultured tobacco cells.

Molybdate and selenate are structural analogs of sulfate that inhibit synthesis of adenosine 5'-phosphosulfate by ATP sulfurylase (sulfate adenylyltransferase, ATP:sulfate adenylyltransferase, EC 2.7.7.4) in crude extracts of tobacco XD cells. Both of these anions derepress ATP sulfurylase in cells growing on sulfate, but not in cells growing on L-cysteine. However, the two anions appear to derepress by different mechanisms. Molybdate caused derepression only at concentrations that were in excess over sulfate and were sufficient to inhibit growth and protein accumulation, indicating that the derepression resulted from sulfur starvation. Selenate caused derepression at one-tenth the concentration of sulfate, a concentration of selenate that was subtoxic, while toxic levels of selenate produced far less derepression. The susceptibility of the tobacco cells to selenate toxicity was high under conditions of sulfur nutrition that derepress ATP sulfurylase, and low under conditions that repress ATP sulfurylase, in agreement with the idea that selenate acts via a functional sulfate assimilation pathway. Since it is known that selenate is incorporated into analogs of sulfur compounds, it is proposed that the tobacco cells synthesize the seleno-analog of the end product of the sulfate pathway responsible for repression, and the seleno-analog antagonizes the normal end product in the repression mechanism, the net result being derepression of ATP sulfurylase by selenate.

Control of cell division in Saccharomyces cerevisiae by methionyl-tRNA.

We suggest that two events are necessary for an asynchronous population of cells to undergo arrest in the GI phase of the cell cycle upon nutrient starvation. First, passage through GI must be prevented by a deficiency of some metabolic intermediate. Since this intermediate may act indirectly to arrest division, we designate it the "signal". We have found three conditions under which Saccharomyces cerevisiae cells arrest division in GI: sulfate starvation of a prototroph, methionine starvation of an auxotroph, or a shift of a conditional methionyl-tRNA synthetase mutant [L-methionine: tRNA Met ligase (AMP-forming), EC 6.1.1.10] to a restrictive condition. We interpret these results to indicate that the signal for sulfate starvation in S. cerevisiae is generated near the end of the sulfate assimilation pathway (at or beyond the formation of mehtionyl-tRNA). As a unifying hypothesis, we propose that the signal for all nutrients is generated at the level of protein biosynthesis.

Regulation of S-amino acids biosynthesis in Aspergillus nidulans. Role of cysteine and-or homocysteine as regulatory effectors.

It was found that in Aspergillus nidulans the enzymes of the sulfate assimilation pathway and O-acetylhomoserine sulfhydrylase are under cysteine- and/or homocysteine, but not methionine- or S-adenosylmethionine-mediated regulation. These enzymes are repressed when the cells are grown in the presence of cysteine or homocysteine even in conditions where cysteine cannot be a precursor of homocysteine and vice versa. This was demonstrated by using mutants with impaired cystathionine cleavage enzymes. Thus, these two amino acids can substitute each other as the regulatory effectors. The addition of methionine causes repression only in conditions when it can be metabolized to homocysteine. The mutant cysA1 with a block at the serine transacetylase step is a prototroph owing to the existence of an alternative pathway for cysteine synthesis involving the enzymes: homocysteine synthase, cystathionine β-synthase and γ-cystathionase. All the three enzymes as well as those of the sulfate assimilation pathway are derepressed in this mutant. CysA1 mutation supresses the meth55 mutant blocked at β-cystathionase owing to the derepression of homocysteine synthase, so that the cystathionine cleavage step is bypassed. The results indicate that the pathway involving cystathionine formation is the main one for methionine biosynthesis in A. nidulans. The pathway involving homocysteine synthase is an alternative-conditional one, physiologically effective only when this enzyme is derepressed.

Regulation of sulfate transport in neurospora by transinhibition and by inositol depletion

Neurospora possesses two distinct sulfate transport systems, a low-affinity form (Permease I) which is the only type found in conidia, and a second species (Permease II) which predominates during the mycelial stage. Although methionine represses the synthesis of both of these permeases, inorganic sulfate only partially represses the mycelial form and does not affect the synthesis of Permease I. Both transport systems are also regulated by transinhibition. The transinhibition which occurs in mycelia is not due to an intracellular pool of inorganic sulfate, but is instead exerted by an early intermediate of the sulfate assimilatory pathway. The development of functional sulfate transport activity depends upon genetic and metabolic events which affect the cell membrane. The synthesis of sulfate permease activity in the inos mutant requires an exogenous supply of inositol. The effect of the cot mutant, which is thought to interfere with membrane synthesis, also prevents the development of sulfate permease at the restrictive temperature. The maintenance of pre-existing functional sulfate permease activity apparently also requires a continuous renewal of membrane components since withdrawal of inositol from inos mutants results in a rapid inactivation of transport activity.

Studies on yeast sulfite reductase II. Partial purification and properties of genetically incomplete sulfite reductases

Enzymes catalyzing the reduction of sulfite by reduced methyl viologen (MVH) were partially purified from four mutants of Saccharomyces cerevisiae, strains 6, 11, 20 and 21, which are genetically blocked in the sulfite reduction step in the sulfate assimilation pathway. Unlike NADPH-sulfite reductase from the wild-type strain, the enzymes from the mutants showed no activities coupled to NADPH oxidation. Sedimentation coefficients of these mutant enzymes ranged from 5.1 S to 6.6 S, values which are much smaller than the value of 14.8 S determined for yeast NADPH-sulfite reductase. All the mutant enzymes contained a chromophore or chromophores absorbing at 386 and 587 mμ. In contrast to the wild-type enzyme possessing both FMN and FAD, the enzymes from strains 6, 11 and 20 contained only FMN, and that from strain 21 lacked both flavins. Iron and acid-labile sulfide were detected in these mutant enzymes as well as in the wild-type enzyme.