Short description about sulphur metabolism.

1. WELCOME
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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
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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 then3

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
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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…
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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 (So4
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+/So4
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.
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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.
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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.
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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
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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.
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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.
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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.
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14. Sulphate Reduction
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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
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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.
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17. Regulatory pathway of sulphur metabolism
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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.
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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.
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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
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21. Homocysteine is converted to methionine, catalyzed by the
enzyme methionine synthase
THF- Tri hydro folate
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22. O-Succinylhomoserine
Fig:- Methionine synthesis
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23. Enzymes involved in methionine biosynthesis:
1. aspartokinase
2. ß-aspartate semialdehyde dehydrogenase
3. homoserine dehydrogenase
4. homoserine O-transsuccinylase
5. cystathionine-γ-synthase
6. cystathionine-ß-lyase
7. methionine synthase (in mammals, this step is
performed by homocysteine methyltransferase)
8. Methyl transferase
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24. Methionine Degradation
Methyl
transferase
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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.
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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.
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27. Cysteine biosynthesis
 In animals cysteine is synthesized from homocysteine, a produce of the essential
amino acid methionine.
+ + H2O
+ H2O + + NH3
cystathionine
cystathionine cysteine
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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.
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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.
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30. Fig: cysteine Biosynthesis pathway.
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31. Cysteine Degradation
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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 So4
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.
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33. Reduced Glutathione
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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.
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35. 35

36. Ecological significance of H2S
emissions by plants
 The emission of several volatile reduced sulfur gases (H2S, COS, DMS, CS2 and
methylmercaptan ) from various plant species was determined in various
experiments.
 From these volatile substances H2S is one of the most important sulfur gases
emitted by higher plants in response to an excess of sulfur.
 Soil applied sulfur fertilization and H2S 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.
 H2S 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.
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37. SO2 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
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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.
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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
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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
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