This presentation includes: 1: Introduction 2: Biological nitrogen Fixation- Asymbiotic and symbiotic 3: Mechanism of Nitrogen Fixation 4: Nitrate reduction 5: Ammonia assimilation 6: Nif genes This presentation is prepared for the teachers and students of life sciences (Biology). it will also help for those who are interested in NET/ SLET and competitive examinations.

1. skhot1976@gmail.com Nitrogen Metabolismskhot1976@gmail.com
U.1 Enzymes and Nitrogen Metabolism
1.b NITROGEN METABOLISM
Dr. Sudhakar Sambhaji Khot
M.Sc., Ph.D., SET
Assistant Professor in Botany
Y. C. Warana Mahavidyalaya, Warananagar
SEMESTER- IV
Botany Paper VIII: DSC D14: PLANT METABOLISM

2. skhot1976@gmail.com Nitrogen Metabolism
SEMESTER- IV
Botany Paper VIII: DSC D14: PLANT METABOLISM
CREDITS: 2, LECTURE PERIOD: 3 PER WEEK- LECTURE HOURS
2.4 PER WEEK, MARKS: 50
Dr. S. S. KHOT 2
1.b: Nitrogen
Metabolism
1.7: Introduction
1.8: Biological nitrogen Fixation-
Asymbiotic and symbiotic
1.9: Mechanism of Nitrogen Fixation
1.10: Nitrate reduction
1.11: Ammonia assimilation
1.12: Nif genes
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INTRODUCTION
• Definition
– Assimilation (synthesis and breakdown) of nitrogen containing
compounds in plants is called nitrogen metabolism
• Nitrogen is one of the macro elements, required in higher
concentration by plants
• It is component of nucleic acids (DNA and RNA)
• It is component of enzymes and proteins
• Essential for normal cell division and protein synthesis (growth)
• Component of photosynthetic pigments
• Component of energy carrying molecules i.e. ATP, GTP etc.
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Explains, why plants are supplied with nitrogenous fertilizers for better growth?

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Biogeochemical Nitrogen cycle
• Important processes
– Nitrogen fixation
• Biological, photo-chemical
– Nitrate reduction
• Nitrate→Nitrite→Ammonia
– Nitrification
• Ammonia →Nitrate
– Ammonification
• Organic nitrogen →ammonia
– Denitrification
• Nitrate →N2
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5. skhot1976@gmail.com Nitrogen Metabolism
Biological Nitrogen Fixation
• Definition: Conversion of molecular nitrogen into ammonia by
microorganisms (living organisms) is known as biological nitrogen fixation
• It contributes about 90% of N2 fixed on earth
• @ normal atm pressure and 200C temp, enzyme nitrogenase
• N2 fixing microbes are called diazotrophs
• Two types:
– Symbiotic N2 fixation
– Non-symbiotic N2 fixation
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Biological Nitrogen Fixation: Symbiotic
• By symbiotic diazotrophs
• Forming root nodules (nodulation)
– Rhizobium with leguminous plants
– Frankia with Casuarina, Alnus
• Without formation of root nodules
– Beijerinckia with sugarcane
– Azotobacter with monocots like wheat, corm, sugarcane etc
• They are host specific
–
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7. skhot1976@gmail.com Nitrogen Metabolism
Biological Nitrogen Fixation: Symbiotic
Root nodulation process
• Host secrete lecithin to attract specific
symbiont
• Rhizobium secretes cytokinin and
polymixin-B
• Causes curling of root hair and infection
thread, Rhizobium passes to cortical
region (bacteroid)
• Infection thread branches and secrete
growth harmone to induce cell division
• Continuous cell division forms root
nodule
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Biological Nitrogen Fixation: Symbiotic
Nitrogen fixing mechanism
• By, Enzyme Nitrogenase: It is sensitive to oxygen, LHb O2 carrier, Enzyme
Hydrogenase
Nitrogenase complex = dinitrogen reductase + dinitrogen nitrogenase
Dinitrogen reductase: smaller subunit, Fe-protein, transfers electron to reduce
larger subunit
Dinitrogen nitrogenase: larger subunit, Mo-Fe protein, reduces nitrogen by adding
electrons and Hydrogen
• Requirements: NADPH2 and ferredoxin, Nitrogenase Complex (Fe-Protein and Mo-
Fe protein), Mg++, ATP, N2
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9. skhot1976@gmail.com Nitrogen Metabolism
Biological Nitrogen Fixation: Symbiotic
Nitrogen fixing mechanism
• By, Enzyme Nitrogenase
• It is sensitive to oxygen
– LHb – O2 carrier
– Enzyme Hydrogenase
• Requirements
NADPH2 and ferredoxin,
Nitrogenase Complex (Fe-
Protein and Mo-Fe protein),
Mg, ATP, N2
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10. skhot1976@gmail.com Nitrogen Metabolism
Biological Nitrogen Fixation: Symbiotic
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Nitrogen → diamide → hydrazine → Ammonia
N2 + 8H+
+ 8e-
+ 16ATP→ 2NH3 + H2 + 16 ADP + 16 iP (4 ATP for 2 electrons)

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Biological Nitrogen Fixation: Asymbiotic
It is nitrogen fixation by free living microbes.
Free living autotrophs
• Aerobic: Oscillatoria, Anabaena, Nostoc
• Anaerobic: Chlorobium vibriforme, Chromatium etc.
Free living heterotrophs
• Clostridium, Azotobacter, Klebsiella pneumoniae
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Biological Nitrogen Fixation: Asymbiotic
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Nitrogen fixing mechanism
• By, Enzyme Nitrogenase
• Requirements
NADPH2 and ferredoxin,
Nitrogenase Complex (Fe-
Protein and Mo-Fe protein),
Mg, ATP, N2

13. skhot1976@gmail.com Nitrogen Metabolism
Nitrate reduction
• Nitrates, produced by photochemical N2 fixation,
• They are available in soil .
• They are water soluble and available to plants.
• Nitrates absorbed by roots.
• In root cell cytoplasm, nitrates are converted into ammonia.
• The process is called nitrate reduction or assimilation.
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14. skhot1976@gmail.com Nitrogen Metabolism
Nitrate reduction
It is stepwise process
1. Conversion of Nitrate to nitrite in cytoplasm
2. Conversion of nitrite to ammonia in plastids (in root or leaves)
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Nitrite → Hypo.Nitrite → Hydroxy-amine → Ammonia

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Ammonia Assimilation
• Ammonia (NH3) synthesized by nitrogen
fixation, nitrate reduction and
ammonification is available for plants
• Excess ammonia is harmful for plant cell
• It adversely affects nitrogenase activity,
energy metabolism and ETC
• Therefore, after absorption of ammonia,
it is rapidly converted to amino acids
• This process is known as ammonia
assimilation
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16. skhot1976@gmail.com Nitrogen Metabolism
Ammonia Assimilation
A) Reductive amination
a. Conversion of α-keto-glutarate into glutamate by addition of ammonia
 It is catalyzed by enzyme glutamate dehydrogenase (GDH)
 Conversion of glutamate to glutamine by glutamine synthetase (GS)
 Conversion of glutamine to glutamate (substrate for transamination) by GOGAT enzyme
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Ammonia Assimilation
B) Transamination
Transfer of amino group from one compound to other is called transamination
As a donar of amino group- glutamate can produce 17 types of amino acids
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18. skhot1976@gmail.com Nitrogen Metabolism
• Amide Synthesis: excess nitrogen is stored in form of metabolic
nitrogen reservoir. i.e. glutamine and asparagine
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Nif Gene
• Present in free living and symbiotic nitrogen fixing bacteria
• May be associated with ‘Nod gene’ and ‘Hup gene’
• Nif gene are present on bacterial chromosome and mega plasmid
• Nif gene serves for nitrogen fixation
• They are well studied in Klebsiella pneumoniae
• In K. pneumoniae: nif gene located between histidine operon and Shikimic
acid operon
• Consist of 7 operons and 17 (20) genes with 24 kb size
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Nif Gene
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Thank You
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