Sulphur metabolism

Short description about sulphur metabolism.

1. WELCOME 1

2. Content Introduction Sulphur uptake & Transport Sulphur Activation Sulfate Assimilation Pathway Sulphate Reduction Regulation of sulphur metabolism Biosynthesis of Glutathione Sulphur Di-oxide toxicity in air Recent Work 2

3. Introduction Sulfur is an essential macronutrient required for plant growth. It is primarily used to synthesize cysteine , methionine and numerous essential and secondary metabolites derived from these amino acids. Sulfate enters a plant primarily through the roots by way of an active uptake mechanism. Gaseous sulfur dioxide readily enters the leaves, where it is assimilated. To reach the chloroplasts, where most of the reduction to sulfide takes place, a sulfate molecule must traverse at least three membrane systems: the plasma membrane of root cell at the soil-plant interface, the plasma membranes of internal cells involved in transport, and the chloroplast membranes. Sulfate is assimilated into organic molecules in one of two oxidation states. Most sulfur is reduced to sulfide by the multistep process. Sulfide is then incorporated into cysteine , becoming the thiol group. The bacteria from Thiobacillus species- Rhodanese & sulfogens are sulfate or thiosulfite solubilising bacteria. 3

4. Sulfate uptake and transport The phenotype of sulfate starvation typically consists of pale-green young leaves, while mature leaves remain dark-green. This phenomenon is different from the symptoms of nitrogen and phosphate deficiency, where young leaves remain green due to support by degrading mature leaves, and has been interpreted as an inefficient activation of cellular sulfate pools. Plants can also metabolize sulfur dioxide taken up in the gaseous form through their stomata. Nonetheless, prolonged exposure (more than 8 hours) to high atmospheric concentrations (greater than 0.3 ppm ) of SO2 causes extensive tissue damage because of the formation of sulfuric acid. Sulfur assimilated in leaves is exported via the phloem to sites of protein synthesis (shoot and root apices, and fruits) mainly as glutathione 4

5. The expression patterns of the three genes nicely fit assumptions summarized from earlier observations: the high affinity transporters are root-specific and inducible by exogenous sulfate defficiency , while the low affinity transporter mRNA is less abundant, but present in roots and leaves. The latter form is only slightly inducible in roots and is repressed in leaves upon sulfate deprivation , suggesting a function in cell-to-cell or intracellular transport. It will be interesting to see whether other types of sulfate transporters exist and to investigate the molecular basis of such essential processes as root uptake , vascular loading and unloading, and intracellular transport of sulfate. The identification of the metabolites and the signal transduction pathway that mediate the transcriptional and post-transcriptional induction and repression of sulfate uptake in the observed tissue-specific and developmental pattern will be crucial for understanding the regulation of sulfate assimilation. Continue… 5

6. Fig: Subcellular compartmentation of major reactions and compounds of sulfur metabolism in a typical plant cell from an autotrophic source tissue . Membrane transport processes (open circles) are indicated in their main direction attributed to this cell type. 6

7. Plasma Membrane Transport Anionic sulfate (So 4 2- ) is relatively abundant in the environment and serves as the primary sulfur source for plants. It is actively transported into roots where it can remain or be distributed to other sites. Transport into cells is mediated by plasma membrane–localized H + /So 4 2- co-transporters that are driven by the electrochemical gradient established by the plasma membrane proton ATPase . Seven genes encoding sulfate transporters have been identified from Arabidopsis Thaliana. 7

8. Transport Into Plastids Sulfate must be transported into plastids where reduction and most of assimilation takes place. Sulfide or thiosulfide are probably exported from plastids since isoenzymes for cysteine synthesis, but not for sulfate reduction, are localized outside of plastids. The nature of the plastid sulfate transporter has been the subject of much speculation but it has not been conclusively identified. 8

9. Sulfate activation Inorganic sulfate is chemically very stable and therefore has to be activated prior to reduction to sulfate or esterification with stable organic compounds. The enzyme ATP- sulfurylase catalyzes the formation of adenosine 5’- phosphosulfate (APS), an energy-rich mixed anhydride of phosphate and sulfate. Adenosine5’-phosphosulfate is an efficient inhibitor of ATP- sulfurylase , but the physiological significance of this possible feed-back mechanism is unclear , since APS is labile at cellular pH and its production is kinetically unfavourable, as indicated by its equilibrium constant ( Keq ≈ 10 -7 ). To achieve substantial product formation the equilibrium has to be pulled forward, but inorganic pyrophosphatase hydrolysis of the product Pyrophosphate ( PPi ) alone is not sufficient. 9

10. Sulfur is among the most versatile elements in living organisms (Hell 1997). Disulfide bridges in proteins play structural and regulatory roles. Sulfur participates in electron transport through iron–sulfur clusters. The catalytic sites for several enzymes and coenzymes, such as urease and coenzyme A, contain sulfur. Secondary metabolites (compounds that are not involved in primary pathways of growth and development) that contain sulfur range from the rhizobial Nod factors antiseptic alliin in garlic and anticarcinogen sulforaphane in broccoli. The versatility of sulfur derives in part from the property that it shares with nitrogen multiple stable oxidation states. SULFUR ASSIMILATION 10

11. Consumption of APS by high affinity enzymes is required to pull substrate flow, either by channelling into sulfate reduction, or by a second ATP-dependent activation which results in 3’-phosphoadenosine 5’-phosphosulfate (PAPS) and is catalyzed by APS- kinase . 3’-Phosphoadenosine 5’-phosphosulfate serves as the preferred donor in sulfate- transfer reactions for esterification of hydroxyl residues, hence animals are able to synthesize PAPS as well, e.g. as donor in tyrosine- sulfation reactions. These organisms circumvent the thermodynamic implications of sulfate activation using a bifunctional PAPS- synthetase to channel the intermediate APS. Recent cloning of cDNAs from a marine worm, Urechis caupo , and rat revealed the existence of polypeptides with structural homology to APS- kinase at their N-terminus and to ATP- sulfurylase at their C-terminus. A similar association of independent proteins may exist in plants. 11

12. Sulfate reduction in plants:- The long-held dogma concerning sulfate reduction stated that, in plants, APS is the substrate for this reaction and the enzyme APS sulfotransferase ( APSSTase ). For a number of reasons, however, the APS pathway in plants was not universally accepted despite years of research, definitive evidence for the existence of APSSTase was not forthcoming. One report of its purification from Euglena gracilis was confounded by the unreasonably low specific activity of the pure enzyme. Subsequently, another group reported that, in vitro, plant APS kinase displays APS sulfotransferase activity as a side reaction. This result, combined with several physical similarities, prompted the idea that perhaps APS sulfotransferase is a kinetic object. 12

13. In contrast to this uncertainty, it is widely believed that prokaryotes (including cyanobacteria ) and fungi use PAPS(3’-phosphoadenosine 5’-phosphosulfate) for sulfate reduction via the enzyme PAPS reductase . Recently, however, direct evidence was published by three independent groups. confirming the existence of an APS-dependent pathway in plants. 13

14. Sulphate Reduction 14

15. As reflected by its name, APSSTase was proposed to catalyze the transfer of sulfate from APS to a thiol acceptor molecule forming a thiosulfonate . The physiological acceptor was envisaged to be glutathione, primarily because it is an efficient substrate and is the most abundant thiol in the chloroplast stroma in which APSSTase is localized. The algal enzyme shows a kinetic constant for glutathione of 0.6 mM, a value that is well below the physiological concentration in the stroma , reported to be between 3 and 10 mM. This is a key finding that supports much of the earlier work on this enzyme. For example, it has long been known that sulfite production from sulfate. Kanno isolating the enzyme from a marine macroalga by maintaining high levels of ammonium sulfate throughout the purification procedure. The lability of APSSTase and that it could be stabilized with high concentrations of sulfate salts , but this finding was never incorporated into a purification scheme. APS sulfotransferase 15

16. Regulation of Sulphur metabolism A. Rate-Limiting Steps in S Pathways Sulfate assimilation is regulated by S status. When the amount of S in the plant is low, many enzymes involved in S acquisition and reduction are up-regulated, including sulfate permease , ATP sulfurylase and APS reductase . Expression of the gene encoding APS reductase is most closely correlated with S status and this enzyme is suspected to be a rate-controlling enzyme for the pathway. There is also indication that ATP sulfurylase may be limiting for sulfate uptake and assimilation, because over-expression of the gene resulted in higher plant levels of both reduced and total S. 16

17. Regulatory pathway of sulphur metabolism 17

18. Another potentially limiting enzyme for Cys formation may be serine acetyltransferase , because over- expresion in cytosol and plastids resulted in 3-fold and 6-fold higher Cys levels, respectively. The regulatory enzyme for GSH synthesis under unstressed conditions is thought to be γ- Glutamylsynthetase . Under metal stress, γ- glutamylsynthetase activity is up-regulated both at the transcription level and the enzyme activity level, and GSH synthetase may become co-limiting. B. Regulation of S Metabolism in Response to the Environment As mentioned above, S limitation induces sulfate uptake and assimilation at the transcriptional level, with GSH as an important signal molecule . While uptake and reduction of S are enhanced under S limitation, the synthesis of secondary S compounds (e.g. sulfation ) is down-regulated, and secondary S compounds such as glucosinolates are even broken down to provide S for essential compounds. 18

19. Sulfur limitation also affects the expression of seed storage proteins the rate of photosynthesis and protein turnover . Conversely, when photosynthesis is reduced , sulfate assimilation is reduced as well. Accumulation of AMP and ADP were reported to inhibit ATP sulfurylase , offering a partial explanation of the mechanism involved. The S assimilation pathway is also regulated in coordination with nitrogen (N) assimilation and the ratio of reduced S to reduced N is typically maintained at 1:20. Reduced S compounds activate the key enzyme of N reduction, nitrate reductase . Similarly, reduced N compounds stimulate the key enzymes of S reduction, ATP sulfurylase . 19

20. Methionine is generally regarded as a member of the aspartate family of amino acids however, most of the metabolic functions of methionine are connected with its sulfur moiety . Prime examples are the role of S- adenosylmethionine (SAM) in numerous methyl-transfer reactions and the observation that the cycle underlying ethylene biosynthesis essentially recovers the methylthio group , but not the ammonia and carbon backbone of methionine. Cystathionine - ɣ - synthase is exclusively plastid localized and catalyzes the first committed step of methionine synthesis, the formation of cystathionine from O- phosphohomoserine and cysteine . Regulation of methionine synthesis is connected to the other routes of the aspartate family at the metabolic level . At the branch point of the pathways, threonine synthase requires SAM as an allosteric activator also acts as an inhibitor of aspartate kinase at the entry of the pathway. Methionine biosynthesis 20

21. Homocysteine is converted to methionine, catalyzed by the enzyme methionine synthase THF- Tri hydro folate 21

22. O- Succinylhomoserine Fig:- Methionine synthesis 22

23. Enzymes involved in methionine biosynthesis: aspartokinase ß- aspartate semialdehyde dehydrogenase homoserine dehydrogenase homoserine O- transsuccinylase cystathionine - γ - synthase cystathionine -ß- lyase methionine synthase (in mammals, this step is performed by homocysteine methyltransferase ) 8. Methyl transferase 23

24. Methionine Degradation Methyl transferase 24

25. Methionine Biosynthesis Inhibitior L- Propargylglycine produced growth Inhibition of exogenous methionine and cystathionine γ - synthase activity. L- Aminoethoxyvinylglycine also produced growth inhibtion and morphological change partially preventable by exogenous methionine. L- Aminoethoxyvinylglycine impairs the cleavage of cystathionine to homocysteine . 25

26. Recycling of methionine SAM is used in a wide variety of biological reactions and represents a major pathway of methionine metabolism. The flux through methionine was analysed in Lemna and it was determined that over 80% of methionine is metabolised into SAM , of which approximately 90% is used for transmethylation reactions . The product of these methylation reactions in higher plants is S- adenosylhomocysteine . is recycled to homocysteine by adenosylhomocysteinase . prior to the re incorporation of a methyl group by methionine synthase and regeneration of the methionine molecule. 26

27. Cysteine biosynthesis In animals cysteine is synthesized from homocysteine , a produce of the essential amino acid methionine. + + H 2 O  + H 2 O  + + NH 3 cystathionine cystathionine cysteine 27

28. In the absence of dietary methionine , animals cannot make cysteine . Bacteria and plants however produce cysteine by a different biosynthetic route. The sulfur used to produce cysteine originates from inorganic sulfur taken up from the environment as sulfate. The inorganic sulfur in sulfate is activated by forming a sulfated ADP analog , PAPS. The sulfur is reduced and released to form sulfite and further reduced to form sulfide (S2-) . The carbon backbone in cysteine is derived from serine. Serine is activated through acetylation by serine acetyltransferase . The acetyl group is then exchanged with a sulfur from sulfide to create cysteine . From cysteine other sulfur containing molecules are synthesized, including methionine. 28

29. Since cysteine is an essential amino acid, manipulation of cysteine content in transgenic plants may be a valuable means of increasing the nutritional content of agricultural plants. Manipulation of enzymes such as serine acetyltransferase may provide one strategy to do this. In a related experiment, bacterial genes for cysteine biosynthesis were engineered into sheep in an attempt to improve wool production. Inhibitors Serine acetyltransferase catalyzes the formation of O- acetylserine from L-Ser and acetyl- CoA in plants and bacteria. In plants, two types of SATase have been described. It act as cysteine feedback inhibitor. One is allosterically inhibited by L- Cys , and the second is not sensitive to L- Cys inhibition. 29

30. Fig: cysteine Biosynthesis pathway. 30

31. Cysteine Degradation 31

32. Biosynthesis of glutathione GSH ( y- glutamyl - cysteinyl - glycine ) and GSSH , reduced and oxidized forms of glutathione, respectively, are readily interchangeable. This tripeptide ( y - Glu-Cys-Gly ) is the dominant non-protein thiol in plants and can play a role in regulating the uptake of So 4 2- by plant roots. It is also a substrate for GSH-S- transferases , which are important for detoxification of xenobiotics , and is the precursor of phytochelatins , peptides that enable plant cells to cope with heavy metals in the environment. GSH is an abundant antioxidant in cells and supports redox buffering . The synthesis of GSH occurs in plastids by a two-step reaction catalyzed by y - glutamylcysteine synthetase and GSH synthetase , genes encoding both have been isolated from Arabidopsis. 32

33. Reduced Glutathione 33

34. Exposure of plants to cadmium induces phytochelatin synthesis. This heavymetal chelator is synthesized from GSH by phytochelatin synthase and consists of repetitions of the y - glutamylcysteine dipeptide that terminates with a glycine . Mutants defective in phytochelatin synthesis are sensitive to heavy metals whereas overexpression of y - glutamylcysteine synthetase or GSH synthetase in Brassica juncea allowed increased cadmium tolerance. Glutathione accumulates after excess feeding of sulfur compounds if the normal regulatory control mechanisms are circumvented , suggesting that glutathione functions as a storage pool for excess cysteine . GSH is synthesized by a γ- glutamyl-cysteine synthase and has been characterized from Nicotiana tabacum . This compound is condensed with glycine by the glutathione synthase , forming GSH. 34

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36. Ecological significance of H 2 S emissions by plants The emission of several volatile reduced sulfur gases (H 2 S, COS, DMS, CS2 and methylmercaptan ) from various plant species was determined in various experiments. From these volatile substances H 2 S is one of the most important sulfur gases emitted by higher plants in response to an excess of sulfur . Soil applied sulfur fertilization and H 2 S emission of agricultural crops was not proven, but it was shown in field experiments that sulfur fertilization and the sulfur nutritional status, respectively had a significant effect on fungal infections in oilseed rape. These findings underline the concept of sulfur-induced resistance (SIR) of plants. H 2 S is highly fungi toxic and therefore a relationship between increasing hydrogen sulfide emissions of plants and a higher resistance of crops against pests and diseases can be assumed. 36

37. SO 2 Toxicity in plants Major sources of sulfur dioxide are coal-burning operations, especially those providing electric power and space heating. Sulfur dioxide emissions can also result from the burning of petroleum and the smelting of sulfur containing ores. Sulfur dioxide enters the leaves mainly through the stomata and the resultant injury is classified as either acute or chronic . Acute injury is caused by absorption of high concentrations of sulfur dioxide in a relatively short time. The symptoms appear as 2-sided lesions that usually occur between the veins and occasionally along the margins of the leaves . The colour of the necrotic area can vary from a light tan or near white to an orange-red or brown depending on the time of year, the plant species affected 37

38. Chronic injury is caused by long-term absorption of sulfur dioxide at sub-lethal concentrations. The symptoms appear as a yellowing or chlorosis of the leaf, and occasionally as a bronzing on the under surface of the leaves. Some crop plants are generally considered susceptible to sulfur dioxide: alfalfa, barley, buckwheat, clover, oats, pumpkin, radish, rhubarb, spinach, squash, Swiss chard and tobacco. Fig:- Acute sulfur dioxide injury to raspberry. the injury occurs between the veins and that the tissue nearest the vein remains healthy. 38

39. Recent Work On Sulphur Metabolism Metabolic control of sulphate uptake & assimilation . A series of feedback loops are proposed in which cellular concentration of pathway to repress or activate expression of genes encoding the protein controlling some of the individual steps in pathway. ATP sulfurylase ATP reductase Sulfite reductase OAS Thiol lyase Serine acetyltransferase 39

40. In addition there is also allostearic regulation of serineacetyltransferase ( SATase ) by o- acetylserine (OAS) & cysteine solid line represent metabolic fluxes,grey lines are feedback control loop. The state of knowledge has been significantly influenced by the isolation of genes for each of the metabolic steps, but not all areas have benefited equally from molecular methods. There is still a clear opportunity for applying gene-cloning methods to learn more about how glutathione and glutathione S-conjugates are transported and degraded. Although significant progress has been made toward elucidating the structure, organization, and regulation of GSTs, the in vivo catalytic function of most GSTs is unknown. It has become increasingly apparent that sulfation reactions play a critical role in controlling developmental signals, but this process is still poorly understood; only a few systems have been described. There has been significant progress in defining through genetics the signaling pathway that regulates sulfur response in Chlamydomonas . 40

41. Thank You 41