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Nitrogen fixation and assimilation by plants and bacteria

What purpose does nitrogen fixation and assimilation serve in the biosphere?Nitrogen fixation takes place in bacteria and is the primary process by which atmospheric N2 gas is converted to ammonia (NH4

+) and nitrogen oxides (NO2-

and NO3-) in the biosphere.

Nitrogen assimilation incorporates this ammonia into amino acids, primarily glutamate and glutamine.

What are the net reactions of nitrogen fixation and assimilation by plants and bacteria?

Nitrogen fixation is mediated by the nitrogenase enzyme complex:

N2 + 8 H+ 8 e- + 16 ATP + 16 H2O 2 NH3 + H2 + 16 ADP + 16 Pi

Nitrogen assimilation using glutamine synthetase and glutamate synthase:

-ketoglutarate + NH4+ + ATP + NADPH + H+

Glutamine + H2O + ADP + Pi + NADP+

Nitrogen fixation and assimilation by plants and bacteria

What are the key enzymes in nitrogen fixation and nitrogen assimilation?

Bacterial nitrogenase complex – is the enzyme that uses redox reactions coupled to ATP hydrolysis to convert N2 gas into 2 NH3.

Glutamine synthetase (GS) - is found in all organisms and it incorporates NH4

+ into glutamate to form glutamine through an ATP coupled redox reaction.

Glutamate synthase (GOGAT) - is found in bacteria, plants, and some insects, and it works in concert with glutamine synthetase to replenish glutamate so that the glutamine synthetase reaction is not substrate limited.

Glutamate dehydrogenase (GDH) - is found in all organisms and it interconverts glutamate, NH4

+, and -ketoglutarate in a redox reaction utilizing either NAD(P)+/NAD(P)H.

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Most plants depend on bacteria to supply nitrogen

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Asimilasi & Reduksi Nitrogen

• Organisme hidup mengandung sejumlah besar nitrogen yang tergabung dalam protein, asam nukleat dan banyak biomolekul lain.

• Nitrat diassimilasi dalam daun dan juga akar• Tanaman Herba assimilasi nitrat pada daun

(meski sering juga terjadi di akar saat awal pertumbuhan)

• Tanaman berkayu (pohon/semak, spt kacang kacang kedelai) assimilasi nitrat terutama dalam akar

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Asimilasi & Reduksi Nitrogen

Selama pertumbuhan organisme autotroph, nitrogen dibutuhkan untuk pembentukan sel dari nitrogen anorganik melalui dua cara:

1.Fiksasi nitrogen dari udara;2.Assimilasi nitrate dari ammonia

yang terkandung dalam air atau tanah.

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SERAPAN N TANAMAN : NO3- & NH4

+

• Bagi tumbuhan yang tidak dapat menambat N2, sumber nitrogen utamanya adalah NO3

- dan NH4+.

• Banyak tanaman yang menyerap nitrogen dalam bentuk NO3

- NH4+

segera dioksidasi menjadi NO3- oleh

bakteri nitrifikasi. • Tapi, komunitas konifer dan rumputan

menyerap sebagian besar nitrogen dalam bentuk NH4

+ sebab nitrifikasi dihambat oleh pH tanah yang rendah atau oleh tanin dan senyawa fenol

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Nitrate assimilation in the roots and leaves of a plant.

• Nitrat harus diubah menjadi NH4

+ di dalam tumbuhan sebelum nitrogen masuk ke asam amino dan senyawa nitrogen lainnya

• Nitrate diambil dari tanah oleh akar.

• Nitrat dapat disimpan sementara dalam vacuola dari sel akar atau direduksi dalam sel epidermis dan cortex dari akar

• Kelebihan nitrat dibawa via pembuluh xylem ke sel mesophyll, dimana nitrate dapat disimpan sementara dalam vacuole.

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• Nitrate direduksi menjadi nitrite dalam cytosol dan kemudian nitrite direduksi lebih lanjut dalam chloroplast menjadi NH4+, dari mana asam amino terbentuk

• NH4+ ini digunakan untuk mensintesis glutamine dan asparagine (yang umumnya dinamakan amida)

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• Dua asam amino (glutamine dan asparagine) dapat dipindahkan ke daun melalui pembuluh xylem.

• Saat kapasitas asimilasi nitrat dalam akar berlebihan nitrat dikeluarkan dari akar ke dalam pembuluh xylem dan terbawa ke daun akibat transpirasi.

• Sejumlah besar nitrat dapat disimpan dalam daun pada vakuola.

• Terkadang penyimpanan vakuola dapat habis karena asimilasi nitrat di siang hari dan terisi lagi pada saat malam hari

• Sebagai contoh, daun bayam memiliki kandungan nitrat tertinggi ditemukan pada pagi hari.

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Tempat asimilasi nitrat ?• Baik akar maupun tajuk memerlukan

senyawa nitrogen organik, tapi pada organ manakah NO3

- direduksi dan digabung dalam senyawa organik?

• Akar beberapa tumbuhan dapat mensintesis semua nitrogen organik yang diperlukan dari NO3

-, sedangkan akar tumbuhan lainnya bergantung pada tajuk untuk memenuhi kebutuhan nitrogen organiknya.

• Proses keseluruhan reduksi NO3- menjadi

NH4+ yang bergantung pada energi dirangkum

pada :• Nitrate dalam mesophyll cells

direduksi menjadi nitrite oleh nitrate reductase yang ada dalam cytosol dan selanjutnya menjadi NH4+oleh nitrite reductase dalam chloroplasts

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Nitrate is reduced to nitrite in the cytosol

• Reaksi-ini-terjadi dalam cytosol di luar setiap organela. • Reduksi nitrat sebagian besar menggunakan NADH sebagai reduktan,

meskipun beberapa tumbuhan yang mengandung nitrate reductase bereaksi dengan NADPH persis sebagaimana NADH.

• Nitrate reductase (NR) pada tanaman tinggi terdiri dari dua sub unit yang identik. The molecular mass of each subunit varies from 99 to 104 kDa, depending on the species.

• Setiap subunit mengandung satu electron transport chain yang terdiri atas satu molekul flavin adenine dinucleotide (FAD), satu heme of the cytochrome-b type (cyt-b557), dan satu cofactor containing molybdenum

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The reduction of nitrite to ammonia proceeds in the plastids

• Reduksi nitrite menjadi ammonia membutuhkan enam elektron• Reaksi ini dikatalisis oleh satu enzim, yaitu: the nitrite reductase ,

yang banyak terdapat dalam plastids. • Enzim ini memanfaatkan reduced ferredoxin sebagai electron donor,

yang disediakan oleh photosystem I sebagai hasil photosynthetic electron transport

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Nitrate assimilation also takes place in the roots

• Asimilasi Nitrate sebagian terjadi, dan pada beberapa species terutama, terjadi dalam akar.

• NH4+ diambil dari tanah yang secara normal difiksasi dalam akar.

• Reduksi nitrate dan nitrite seperti pada fixation of NH4+ dimulai dalam sel akar dengan cara yang sama seperti pada mesophyll cells.

• Tetapi, dalam sel akar diperlukan reduksi yang setara dengan supplied exclusively melalui oksidasi carbohydrates.

• Reduksi nitrite dan selanjutnya fiksasi dari NH4+ terjadi dalam leucoplasts, a differentiated form of plastids

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• ATP yang dibutuhkan untuk glutamine synthesis dapat dihasilkan oleh mitochondria dan dipindahkan ke dalam leucoplasts oleh suatu plastid ATP translocator.

• Glutamate synthase dari leucoplasts juga digunakan mereduksi ferredoxin sebagai redox partner, meskipun beberapa leucoplasts juga mengandung glutamate synthase

• Reduksi nitrate di dalam akar memberikan organic nitrogen compounds terutama dalam bentuk glutamine dan asparagine pada tunas melalui aliran transpirasi dalam pembuluh xylem.

• This is also the case bila NH4+ merupakan sumber nitrogen dalam tanah.

Nitrate assimilation

Nitrate (NO3-) + NADPH + H+ + 2e- NO2

- + H2O + NADP+

(uptake in roots)

Nitrite (NO2-) + 8H+ + 6Fdred + 6e- NH4

+ + 6Fdox + 2H2O(toxic)

Nitrate Reductase

Nitrite Reductase Chloroplasts

Cytoplasm

NH4+ into amino acids (amino group)

Nitrate assimilation

Nitrate (NO3-) + NADPH + H+ +2e- NO2

- + H2O + NADP+

(uptake in roots)

Nitrate Reductase Cytoplasm

Flavin Adenine DinucleotideMolybdenum complex reduces nitrate

2e- (from respiration)2e-

Nitrate assimilationNitrite (NO2

-) + 8H+ + 6Fdred + 6e- NH4+ + 6Fdox + 2H2O

(toxic)

Nitrite Reductase Chloroplasts

Photosynthesis ETC

Iron-sulfur

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The end product of nitrate assimilationis a whole spectrum of amino acids

• Semua asam amino dapat dianggap sebagai produk akhir asimilasi nitrat

• Synthesis asam amino ini terutama berlangsung dalam chloroplasts.

• Pola sintesis asam amino sangatlah beragam tergantung pada spesies dan kondisi metabolisme.

• Dalam banyak kasus glutamate dan glutamine mewakili /menggambarkan bagian utama dari synthesized amino acids.

• Glutamate diekspor dari chloroplasts dalam perubahan malate dan glutamine menjadi glutamate

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• serine dan glycine, yang terbentuk sebagai intermediate products dalam photorespiratory cycle, juga menunjukkan bagian penting dari total asam amino yang ada dalam mesophyll cells.

• Sejumlah besar alanine sering terbentuk dalam C4 plants

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

• CO2 assimilation menyediakan carbon skeletons yang dibutuhkan untuk synthesis bermacam-macam amino acids.

• Gambar disamping menunjukkan ringkasan dari awal carbon skeletons dari individual amino acids.

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• 3-Phosphoglycerate merupakan carbon precursor yang sangat penting untuk synthesis of amino acids.

• Ini dihasilkan dalam Calvin cycle dan diekspor dari chloroplasts ke cytosol melalui triose phosphate-phosphate translocator dalam perubahan phosphate.

• 3-Phosphoglycerate diubah dalam cytosol oleh phosphoglycerate mutase dan enolase menjadi phosphoenolpyruvate (PEP)

• From PEP two pathways branch off, the reaction via pyruvate kinase leading to pyruvate, and via PEP-carboxylase to oxaloacetate.

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Yang berwarna merah adalah:

Carbonskeletons untuksynthesis aminoacids yangDiperolehmelalui asimilasiCO2 MerupakanPrekursorpenting untuksintesis aminoacid

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The synthesis of glutamate requires the participation of mitochondrial metabolism

• Glutamate terbentuk dari α-ketoglutarate, yang dapat diperoleh melalui sekuens terpisah pada siklus asam sitrat di mitokondria.

• Pyruvate and oxaloacetate ditranspor dari cytosol ke mitochondria oleh suatu translocator tertentu.

• Pyruvate dioksidasi oleh pyruvate dehydrogenase menjadi acetyl-CoA yang kemudian berkondensasi dengan oxaloacetate menjadi citrate.

• Citrate ini dapat dikonversi dalam mitochondria melalui aconitase, dioksidasi lebih lanjut oleh NAD-isocitrate dehydrogenase, dan menghasilkan α-ketoglutarate dapat ditranspor ke dalam cytosol oleh specific translocator.

The Fate of Ammonium

• Ammonium enters organic linkage via three major reactions that are found in all cells.

• The enzymes mediating these reactions are (1)Carbamoyl-phosphate synthetase I, (2)Glutamate dehydrogenase (GDH), (3)Glutamine synthetase (GS), and(4)Glutamate synthase (GOGAT).

The Fate of AmmoniumMajor reactions in all cells

• Carbamoyl-phosphate synthetase I– two ATP required - one to activate bicarb, one to

phosphorylate carbamate • Glutamate dehydrogenase (GDH)

– reductive amination of alpha-ketoglutarate to form glutamate

• Glutamine synthetase (GS)– ATP-dependent amidation of gamma-carboxyl of

glutamate to glutamine • Glutamate synthase / GOGAT

– reductive amination of a-keto-glutarate using the amide-N of glutamine as the N donor resulting glutamate

carbamoyl-phosphate synthetase I

• Carbamoyl-phosphate synthetase I catalyzes one of the steps in the urea cycle.

• Two ATP are consumed :(1)in the activation of HCO3

- for reaction with ammonium,

(2)in the phosphorylation of the carbamate

glutamate dehydrogenase

• Glutamate dehydrogenase (GDH) catalyzes the reductive amination of a-ketoglutarate to yield glutamate.

glutamine synthetase

• Glutamine synthetase (GS) catalyzes the ATP-dependent amidation of the gamma-carboxyl group of glutamate to form glutamine.

• The reaction proceeds via a gamma-glutamyl-phosphate intermediate, and GS activity depends on the presence of divalent cations such as Mg2+ (Figure 26.10).

Glutamine

• Glutamine major N donor in the biosynthesis of many organic N compounds such as purines, pyrimidines, and other amino acids.

• The amide-N of glutamine provides the nitrogen atom in these biosyntheses.

• GDH and GS responsible for most of the ammonium assimilated into organic compounds.

Ammonium AssimilationTwo principal pathways

• Principal route: GDH+GS in organisms rich in N • Secondary route: GS+GOGAT in organisms

confronting N limitation • GOGAT is glutamate synthase glutamate:oxo-glutarate amino transferase

• In organisms that enjoy environments rich in nitrogen, GDH and GS acting in sequence furnish the principal route of NH4

+ incorporation GDH generates glutamate & GS use glutamate to form glutamine.

• Green plants which grow under conditions where little NH4+ is available GDH is not effective need another option to generate glutamate which GS can use to form glutamine.

Ammonium Assimilation(2 Principles Pathway)

• Limited availibility of NH4+ creates the need for an

alternative mode of glutamate synthesis to replenish the glutamate consumed by the GS reaction.

• This need is filled by glutamate synthase (also known as GOGAT, the acronym for the other name of this enzyme - glutamate:oxo-glutarate amino-transferase).

• Glutamate synthase catalyzes the reductive amination of a-keto-glutarate using the amide-N of glutamine as the N donor to generate glutamate

Ammonium Assimilation(2 Principles Pathway)

• Two glutamates are formed from amination of a-ketoglutarate and the other from deamidation of Gln

• These glutamates can now serve as ammonium acceptors for glutamine synthesis by GS.

• Organisms variously use NADH, NADPH, or reduced ferredoxin as reductant.

• Together, GS and GOGAT constitute a second pathway of ammonium assimilation, in which GS is the only NH4

+-fixing step; the role of GOGAT is to regenerate glutamate (Figure 26.13).

coping with a nitrogen-limited environment has its cost

• Note that GS/GOGAT pathway consumes two equivalents of ATP and 1 NADPH (or similar reductant) per pair of NH4

+- atoms into Gln.

• In contrast, the GDH/GS pathway, used up only 1 ATP and 1 NADPH per pair of NH4

+- fixed.

Nitrogen assimilation in plantsNitrogen assimilation proceeds in one of two ways.

First, if NH4+ levels in the soil are high, plants can use the glutamate

dehydrogenase (GDH) reaction to directly incorporate NH4+ into the amino

acid glutamate using -ketoglutarate as the carbon skeleton.

Note that most often, this reaction releases NH4+ from glutamate in other contexts.

Nitrogen assimilation in plants

A second, and more common way that plants and bacteria incorporate NH4+

into metabolites, is through a two reaction mechanism that functions when NH4+

concentrations are low. In this mechanism, the enzyme glutamine synthetase (GS) uses ATP in a coupled reaction to form glutamine from glutamate using NH4

+.

Nitrogen assimilation in plants

Next, the glutamine is combined with -ketoglutarate in a reaction catalyzed by the enzyme glutamate synthase (GOGAT) to form two molecules of glutamate (glutamine contains two nitrogens).

The net reaction is nitrogen assimilation

Importantly, the newly acquired nitrogen in glutamate and glutamine is used to synthesize a variety of other amino acids through aminotransferase enzymes such as aspartate aminotransferase.

Biosynthesis of proline and arginine from glutamate in bacteria. All five carbon atoms of proline arise from glutamate. In many organisms, glutamate dehydrogenase is unusual in that it uses either NADH or NADPH as a cofactor.

The γ-semialdehyde in the proline pathway undergoes a rapid, reversible cyclization to Δ1-pyrroline-5-carboxylate (P5C), with the equilibrium favoring P5C formation.

Cyclization is averted in the ornithine/arginine pathway by acetylation of the α-amino group of glutamate in the first step and removal of the acetyl group after the transamination.

Biosynthesis of serine from 3-phosphoglycerate and of glycine from serine in all organisms. Glycine is also made from CO2 and NH4

+ by the action of glycine synthase, with N5,N10-methylenetetrahydrofolate as methyl group donor

Biosynthesis of cysteine from serine in bacteria and plants. The origin of reduced sulfur is shown in the pathway on the right.

Biosynthesis of cysteine from homocysteine and serine in mammals. The homocysteine is formed from methionine.