NITRATE ASSIMILATION(Essential for the Synthesis of Organic Matter)

(CS 701) Advanced Biochemistry

ARNOLD V. DAMASOMS in Crop Science- AgronomyCentral Luzon State University

Nitrate Assimilation(Essential for the Synthesis of Organic Matter)

• Introduction • Living matter contains a large amount of nitrogen

incorporated in proteins, nucleic acids, and many other biomolecules.

• This organic nitrogen is present in oxidation state –III (as in NH3).

• During autotrophic growth the nitrogen demand for the formation of cellular matter is met by inorganic nitrogen in two alternative ways:

1. Fixation of molecular nitrogen from air; or

2. Assimilation of the nitrate or ammonia contained in water or soil.

• Nitrogen assimilation is the formation of organic nitrogen compounds like amino acids from inorganic nitrogen compounds present in the environment. Organisms like plants, fungi and certain bacteria that cannot fix nitrogen gas (N2) depend on the ability to assimilate nitrate or ammonia for their needs.

• Other organisms, like animals, depend entirely on organic nitrogen from their food. Plants absorb nitrogen from the soil in the form of nitrate (NO3

−) and ammonia (NH3). In aerobic soils where nitrification can occur, nitrate is usually the predominant form of available nitrogen that is absorbed

Importance and Significance

The reduction of nitrate to NH3

(Proceeds in two partial reactions)• Nitrate is assimilated in the leaves and also in

the roots. • In most fully grown herbaceous plants, nitrate

assimilation occurs primarily in the leaves, although nitrate assimilation in the roots often plays a major role at an early growth state of these plants. In contrast, many woody plants (e.g., trees, shrubs), as well as legumes such as soybean, assimilate nitrate mainly in the roots.

• The transport of nitrate into the root cells proceeds as symport with two protons (Fig. 1). A proton gradient across the plasma membrane, generated by an H+-P-ATPase drives the uptake of nitrate against a concentration gradient.

• The ATP required for the formation of the proton gradient is mostly provided by mitochondrial respiration

• The nitrate taken up into the root cells can be stored there temporarily in the vacuole. As discussed, nitrate is reduced to NH4+ in the epidermal and cortical cells of the root. This NH4

+ is mainly used for the synthesis of glutamine and asparagine

Figure 2. A. Nitrate reductase transfers electrons from NADH to nitrate. B. The enzyme contains three domains where FAD, heme, and the molybdenum cofactor (MoCo) are bound.

Figure 1. Nitrate assimilation in the roots and leaves of a plant.

Figure 3. The molybdenum cofactor (Moco)

Nitrate is reduced to nitrite in the cytosol

• Nitrate reduction uses mostly NADH as reductant, although some plants contain a nitrate reductase reacting with NADPH as well as with NADH. The nitrate reductase of higher plants consists of two identical subunits. The molecular mass of each subunit varies from 99 to 104 kDa, depending on the species

Figure 4. Nitrate reductase in chloroplasts transfers electrons from ferredoxin to nitrate. Reduction of ferredoxin by photosystem I that shown in figure.

The reduction of nitrite to ammonia proceeds in the plastids

• To a much lesser extent, the ferredoxin required for nitrite reduction in a leaf can also be provided during darkness via reduction by NADPH, which is generated by the oxidative pentose phosphate pathway present in chloroplasts and leucoplasts.

• Nitrite reductase contains a covalently bound 4Fe-4S cluster, one molecule of FAD, and one siroheme. Siroheme (Fig. 5) is a cyclic tetrapyrrole with one Fe-atom in the center. Its structure is different from that of heme as it contains additional acetyl and propionyl residues deriving from pyrrole synthesis

Figure 5. Structure of siroheme

• The 4Fe-4S cluster, FAD, and siroheme form an electron transport chain by which electrons are transferred from ferredoxin to nitrite. Nitrite reductase has a very high affinity for nitrite. The capacity for nitrite reduction in the chloroplasts is much greater than that for nitrate reduction in the cytosol

The fixation of NH4+ proceeds in the same way as in photorespiration cycle

• Glutamine synthetase in the chloroplasts transfers the newly formed NH4

+ at the expense of ATP to glutamate, forming glutamine. The activity of glutamine synthetase and its affinity for NH4+ (Km 510-6 mol/L) are so high that the NH4

+ produced by nitrite reductase is taken up completely. The same reaction also fixes the NH4+ released during photorespiration

Figure 5.1. Reductase of photosystem I

Figure 6. Compartmentation of partial reactions of nitrate assimilation and the photorespiratory pathway in mesophyll cells.

• Glufosinate (Fig. 7), a substrate analogue of glutamate, inhibits glutamine synthesis. Plants in which the addition of glufosinate has inhibited the synthesis of glutamine accumulate toxic levels of ammonia and die off. NH4+-glufosinate is distributed as an herbicide under the trade name Liberty (Aventis).

Figure 7. Glufosinate

Nitrate assimilation (also takes place in the roots)

• As mentioned, nitrate assimilation occurs in part, and in some species even mainly, in the roots. NH4+ taken up from the soil is normally fixed in the roots.

• The reduction of nitrate and nitrite as well as the fixation of NH4+ proceeds in the root cells in an analogous way to the mesophyll cells.

The oxidative pentose phosphate pathway provides reducing equivalents for nitrite reduction in leucoplasts

• The reducing equivalents required for the reduction of nitrite and the formation of glutamate are provided in leucoplasts by oxidation of glucose 6 phosphate via the oxidative pentose phosphate pathway discussed in (Fig. 8).

• As in chloroplasts, nitrite reduction in leucoplasts also requires reduced ferredoxin as reductant. In the leucoplasts, ferredoxin is reduced by NADPH, which is generated by the oxidative pentose phosphate pathway. The ATP required for glutamine synthesis in the leucoplasts can be generated by the mitochondria and transported into the leucoplasts by a plastid ATP translocator in counter-exchange for ADP

Figure 8. The oxidative pentose phosphate pathway

Nitrate assimilation(is strictly controlled)

• During photosynthesis, CO2 assimilation and nitrate assimilation have to be matched to each other. Nitrate assimilation can progress only when CO2 assimilation provides the carbon skeletons for the amino acids.

• Moreover, nitrate assimilation must be regulated in such a way that the production of amino acids does not exceed demand

• Finally, it is important that nitrate reduction does not proceed faster than nitrite reduction, since otherwise toxic levels of nitrite would accumulate in the cells.

• Under certain conditions such a dangerous accumulation of nitrite can indeed occur in roots when excessive moisture makes the soil anaerobic

The synthesis of the nitrate reductase protein is regulated at the level of gene expression

• Nitrate reductase is an exceptionally short-lived protein. Its half-life is only a few hours. The rate of de novo synthesis of this enzyme is therefore very high. Thus, by regulating its synthesis, the activity of nitrate reductase can be altered within hours.

Nitrate reductase is also regulated by reversible covalent modification • The regulation of de novo synthesis of nitrate

reductase (NR) allows regulation of the enzyme activity within a time span of hours.

• This would not be sufficient to prevent an accumulation of nitrite in the plants during darkening or sudden shading of the plant. Rapid inactivation of nitrate reductase in the time span of minutes occurs via phosphorylation of the nitrate reductase protein (Fig. 9).

Figure 9. Regulation of nitrate reductase (NR).

14-3-3 Proteins are important metabolic regulators

• It was discovered that the nitrate reductase inhibitor protein belongs to a family of regulatory proteins called 14-3-3 proteins, which are widely spread throughout the animal and plant worlds.

• 14-3-3 proteins bind to a specific binding site of the target protein with six amino acids, containing a serine phosphate in position 4

The regulation of nitrate reductase and sucrose phosphate synthase have great similarities

• The mechanism of the regulation of nitrate reduction by phosphorylation of serine residues of the enzyme protein by special protein kinases and protein phosphatases is remarkably similar to the regulation of sucrose phosphate synthase.

The end product of nitrate assimilation(is a whole spectrum of amino acids)

• The carbohydrates formed as the product of CO2 assimilation are transported from the leaves via the sieve tubes to various parts of the plants only in defined transport forms, such as sucrose, sugar alcohols (e.g., sorbitol), or raffinoses, depending on the species.

CO2 assimilation provides the carbon skeletons to synthesize the end products of nitrate assimilation

• CO2 assimilation provides the carbon skeletons required for the synthesis of the various amino acids. Figure 10 gives an overview of the origin of the carbon skeletons of individual amino acids.

Figure 10. Origin of carbon skeletons for various amino acids.

Figure 11. Carbon skeletons for the synthesis of amino acids are provided by CO2 assimilation.

The synthesis of glutamate requires the participation of mitochondrial metabolism

• That glutamate is formed from -ketoglutarate, which can be provided by a partial sequence of the mitochondrial citrate cycle (Fig. 11). Pyruvate and oxaloacetate are transported from the cytosol to the mitochondria by specific translocators. Pyruvate is oxidized by pyruvate dehydrogenase, and the acetyl-CoA thus generated condenses with oxaloacetate to citrate.

Biosynthesis of proline and arginine

• proline has a special function as a protective substance against dehydration damage in leaves. When exposed to aridity or to a high salt content in the soil (both leading to water stress), many plants accumulate very high amounts of proline in their leaves, in some cases several times the sum of all the other amino acids

Figure 12. Pathway for the formation of amino acids from glutamate.

Aspartate is the precursor of five amino acids

• Aspartate is formed from oxaloacetate by transamination with glutamate by glutamate-oxaloacetate amino transferase (Fig. 14).

• The synthesis of asparagine from aspartate requires a transitory phosphorylation of the terminal carboxylic group by ATP, as in the synthesis of glutamine. In contrast to glutamine synthesis, however, it is not NH4+ but the amide group of glutamine that usually serves as the amino donor in asparagine synthesis

Figure 13. Two compatible substances that, like proline, are accumulated in plants as protective agents against desiccation and high salt content in the soil.

Figure 14. The pathway for the synthesis of amino acids from aspartate.

Figure 15. Feedback inhibition by end products regulates the entrance enzyme for the synthesis of amino acids from aspartate according to demand. [-] indicates inhibition. Aspartate kinase exists in two isoforms

Acetolactate synthase participates in the synthesis of hydrophobic amino acids

• Acetolactate synthase, catalyzing this reaction, contains thiamine pyrophosphate (TPP) as its prosthetic group. The reaction of TPP with pyruvate yields hydroxyethyl-TPP and CO2, in the same way as in the pyruvate dehydrogenase reaction.

Figure 16A. Pathway for the synthesis of amino acids from pyruvate.

Figure 17. Synthesis of valine and leucine.

Figure 16B. Pathway for the synthesis of isoleucine from threonine and pyruvate.

Figure 18. Herbicides: chlorsulfurone, a sulfonyl urea, (trade name Glean, DuPont) and imazethapyr, an imidazolinone, (trade name Pursuit, ACC) inhibit acetolactate synthase. Glyphosate (trade name Roundup, Monsanto) inhibits EPSP synthase

Aromatic amino acids are synthesized via the shikimate pathway

• Precursors for the formation of aromatic amino acids are erythrose 4phosphate and phosphoenolpyruvate.

• These two compounds condense to form cyclic dehydrochinate accompanied by the liberation of both phosphate groups (Fig. 19)

Figure 19. Aromatic amino acids are synthesized by the shikimate pathway. PEP = phosphoenolpyruvate.

Figure 20. Several steps in the synthesis of aromatic amino acids

A large proportion of the total plant matter can be formed by the shikimate pathway

• The function of the shikimate pathway is not restricted to the generation of amino acids for protein biosynthesis. It also provides precursors for a large variety of other substances (Fig. 21) formed by plants in large quantities, particularly phenylpropanoids such as flavonoids and lignin

Figure 21. Several secondary metabolites are synthesized via the shikimate pathway.

Glutamate is the precursor (for synthesis of chlorophylls and cytochromes)

• Chlorophyll amounts to 1% to 2% of the dry matter of leaves. Its synthesis proceeds in the plastids. Chlorophyll consists of a tetrapyrrole ring with magnesium as the central atom and with a phytol side chain as a membrane anchor. Heme, likewise a tetrapyrrole, but with iron as the central atom, is a constituent of cytochromes and catalase.

Figure 22. In chloroplasts, glutamate is the precursor for the synthesis of -amino levulinate, which is condensed to porphobilinogen.

Protophorphyrin is also a precursor for heme synthesis

• By assembling the heme with apoproteins, chloroplasts are able to synthesize their own cytochromes. Also, mitochondria possess the enzymes for the biosynthesis of their cytochromes from protoporphyrin IX, but the corresponding enzyme proteins are different from those in the chloroplasts.

Figure 23. Protoporphyrin synthesis.

Figure 24 Overview of the synthesis of chlorophyll and heme in chloroplasts.

Summary

• Nitrate assimilation is the formation of organic nitrogen compounds like amino acids from inorganic nitrogen compounds present in the environment. Organisms like plants, fungi and certain bacteria that cannot fix nitrogen gas (N2) depend on the ability to assimilate nitrate or ammonia for their needs.

• Plants like castor reduce a lot of nitrate in the root itself, and excrete the resulting base. Some of the base produced in the shoots is transported to the roots as salts of organic acids while a small amount of the carboxylates are just stored in the shoot itself

• However, about 99% of the organic nitrogen in the biosphere is derived from the assimilation of nitrate. NH4

+ is formed as an end product of the degradation of organic matter, primarily by the metabolism of animals and bacteria, and is oxidized to nitrate again by nitrifying bacteria in the soil. Thus a continuous cycle exists between the nitrate in the soil and the organic nitrogen in the plants growing on it.

• While nearly all the ammonia in the root is usually incorporated into amino acids at the root itself, plants may transport significant amounts of ammonium ions in the xylem to be fixed in the shoots. This may help avoid the transport of organic compounds down to the roots just to carry the nitrogen back as amino acids.

End of Report