Here , we have presented the information on nitrate assimilation, enlisting all the enzymes involved in this process. The nitrogen cycle as well as uses of nitrate has been nicely explained.
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Carbon and Nitrogen Metabolism-Lecture-1 (2).pptx
1. INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI-110012
E-mail: ranjeetranjaniari@gmail.com; ranjeet.kumar@icar.gov.in; Cel No: +91-08368958133
Carbon and Nitrogen Metabolism
BIOCHEM-510
Ranjeet R. Kumar
Senior Scientist
2. Topic for the Lecture
• Nitrogen Cycle
• Biochemistry of nitrate assimilation
• Regulation in nitrate assimilatory pathway
• GS-GOGAT cycle
• Ureides and amides as nitrogen transport compounds
3. Nitrogen
• 78% in the atmosphere
• Most abundant element
• Essential nutrients
• Important component of central metabolism pathway
• Part of various structural component
• 1.5-2% of the dry weight of plant tissue
• Major constraints for crop productivity
4. Nitrate
• Key role in biogeochemical nitrogen cycle in both prokaryotes and
eukaryotes
• Source of N2 for assimilation
• Serve as terminal electron acceptor during anaerobic respiration
(Enteric and sulphate reducing bacteria)
• Denitrification process
Nitrate – Nitrite – Nitric oxide – nitrous oxide – Nitrogen - Atmosphere
• Prokaryotes – Paracoccus denitrificans, Pseudomonads, Thiobacillus
5. Nitrite
• Nitrate gets converted into nitrite
• Nitrite oxidises the Fe of Hb from ferrous to ferric.
• Ferric form unable to accept the oxygen
• Leads to Methemoglobinemia
• Prevalent in Infant
• Also called as Blue baby syndrome
8. INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI-110012
E-mail: ranjeetranjaniari@gmail.com; ranjeet.kumar@icar.gov.in; Cel No: +91-08368958133
Carbon and Nitrogen Metabolism
BIOCHEM-501
Ranjeet R. Kumar
Senior Scientist
Lecture - 2
9. Nitrate Uptake
Predominant form available to the plants
Ammonia
80% of the fertilizer
added to the soil in
this form
NO3
-
Nitrifying bacteria Oxidised
10. Function of Nitrate
Nitrate Signalling molecule
Root morphogenesis
Arresting gametogenesis
Carbon metabolism
adaptation
Shoot to root balance
11. Nitrate Uptake Modals
HCO3-
NO3
-
NO3
-
Organic acids
Amino acids
Proteins
Nitrate reduction
Meristematic
Growth
Amide and asparagine - inhibit
both uptake and reduction of
Nitrate
Glutamine – Inhibit only
reduction
12. Nitrate Uptake Modals
HCO3-
NO3
-
NO3
-
Organic acids
Amino acids
Proteins
Nitrate reduction
Pod filling Stage
Amino acids
• Leaf proteolysis occur
• Amino N in leaf is
transported to pod.
• Phloem enriched with
N-Compound
• Repress Nitrate uptake
13. Nitrate Uptake
• Carrier mediated active processes
• Inducible
• Nitrate themselves induce or activate uptake system
• Uptake system is under feed back control
Uptake of nitrate is controlled by –
1) Nitrate reduction
2) Malate production in shoot [Neutralize alkaline condition created during
Nitrate reduction]
Malate + K+ Transported to root Oxidative decarboxylation
Exchanged for Nitrate external Bicarbonate Ions
14. Nitrate Transporters
• Nitrate transporter is key in controlling the efficiency of nitrogen
assimilation
Nitrate Assimilation
Plasma membrane Envelop of chloroplast
Delimit cytosolic reduction of
nitrate to nitrite
Delimit reduction of nitrite to
ammonia
15. Nitrate Transporters
Nitrate Transporter
Physiological Data
(Substrate affinity/ induction)
Gene Sequence Analysis
HANT LANT
Operated at low external
Nitrate concentration
High external nitrate
concentration
<250 uM nitrate concentration >1 mM nitrate concentration
Have different affinities and
capacity to transport nitrate
Corresponds to H+ dependent
active transport
16. Nitrate Transporters
• Identification of genes encoding NT in eukaryotes started with the
cloning of Aspergillus Crn A gene (1991)
• 112 genes encoding NT have been identified in plants.
Nitrate Transporter
Nrt-1 Nrt-2
NRT 1;1 A. thaliana
(LANT/HANT)
NRT 2;2 Chlamydomonas
(LANT)
Arabidopsis – NRT1: 53 genes; NRT-2: 7 genes
17. Nitrate Transporters
NRT-2 NRT-1
Alos called as NNP family (Nitrate nitrite
porter)
Belongs to POT family (H+ dependent
oligo peptide trasnporter)
Belongs to MFS group (Major facilitator
superfamily)
Sugar transporter, Bac-Drug H+ Antiporter,
Metabolite H+ Symporter
NRT-2 protein has 12 transmembrane domains
arranged in two set of six connected by cytosolic
loop
12 transmembrane domain
A consensus motif is present within 5th
transmembrane domain – signature of NNP
family
Long loop with many charged residue seperating
first 6 TM from second 6 Tm
A-G-W/L-G-N-M-G = Substrate recognition
motif
Short N and C terminal end.
NT
19. INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI-110012
E-mail: ranjeetranjaniari@gmail.com; ranjeet.kumar@icar.gov.in; Cel No: +91-08368958133
Carbon and Nitrogen Metabolism
BIOCHEM-501
Ranjeet R. Kumar
Senior Scientist
Lecture - 3
21. Nitrate/ Nitrite Transport System
Nitrate/ Nitrite Transport System
Sys-I Sys-IV
Sys-II Sys-III
Based on the physiological and molecular studies on mutant strains
defective in several nitrate clustered genes
Normal condition – Sys-I and Sys-II transport nitrate
Sys-III, I – Nitrite entry
Under limiting CO2 and NH3– Sys-IV allow nitrate and nitrite transport
22. Nitrate/ Nitrite
Transport System
• These transport systems are
differentially regulated under C
and N sources.
• Sys-I, II and III optimally
expressed by high CO2; Blocked
by NH3
• Sys-IV – Optimally expressed
under eCO2; not blocked by NH3
• SYS-IV – Activity inhibited by
CO2, Cl and Cl channel
inhibitors
23. Structure and Function of Eukaryotic NAD(P)H
Nitrate Reductase
• NAD(P)H Nitrate reductase (EC.1.6.6.1-3
• Molybdenum containing enzyme
• Present in plants fungus and algae
• Reduction of nitrate to nitrite is irreversible
24. NR
~110 kDa Mol. Wt.
Mo-MPT
Heme -Fe
FAD
Soluble Enzyme
Structure and Function of Eukaryotic NAD(P)H
Nitrate Reductase
• Dimerization is required for the activity
• NR is homo-dimer
• Has 2 active site connected by internal ETC
• Internal ETC - FAD-Heme-Fe-Mo-MPT
• Electron donor is NADH or NADPH or NAD(P)H
25. Prokaryotic and Eukaryotic Nitrate Reductase
Prokaryotic NR Eukaryotic NR
MPT has additional nucleotide MPT has less Nt
2 Pterins are coordinated to Mo 1 Pterins are coordinated to Mo
Membrane bound terminal e-
acceptor
Cytosolic NR
Soluble enzyme in periplasmic
space
Soluble enzyme in cytoplasm
Involved in denitrification Not characterized
Fe4-S4 redox center absent
Simple, small and sluggish Complex, large and efficient
26. Nitrate Reductase Modals
• Homo dimer
• 2 identical subunits
• Joined together by Mo
cofactor
• Subunit mol wt. 110-114 kDa
• Cigar shaped
• Axial ration 14:1
• Sedimentation coefficient –
7.9s
• Activity was first
demonstrated in soybean
• Evans and Nason 1953
• NR has Arginine at its active
site
27. Structure and Function of Eukaryotic NAD(P)H
Nitrate Reductase
NAD(P)H+H+
NAD(P) +
NAD(P)H+H+
NAD(P) +
FAD SH Cyt b557
Cyt b557
FAD SH
MoCC
MoCC
NO3-
NO2-
NO3-
NO2-
Cyt C FMNH2
Diaphorase
NAHD Dehydrogenase
C-Terminal end of NR
30 kDa
Terminal NR
N-terminal End
69 kDa
2e-
2e-
28. Structure of Nitrate Reductase
N-terminal seq
Mo-MPT domain
Dimer interface domain
Hinge-1
Cyb b domain
Hinge 2
FAD Binding domain
NAD(P)H binding domain
29. MoCo
• Also called as MCC
• NR is molybdoenzyme
• MO is bound to low molecular weight peptide
• Called as Pterin (reduced form)
• Readily dissociated by acid or heat treatment
• Mol. Wt. 1000 Dalton
• Inaccessible to attack by Trypsin
Dioxo form of
cofactor
NR
Desulpho
form of MoCo
XDH
Abscisic Acid
Aldehyde
Oxidase
30. Site of action of different inhibitors in NR structure
NAD(P)H+H+
NAD(P) +
NAD(P)H+H+
NAD(P) +
FAD SH Cyt b557
Cyt b557
FAD SH
MoCC
MoCC
NO3-
NO2-
NO3-
NO2-
Cyt C FMNH2
2e-
2e-
P-Chloromercuric benzoic acid
Cyanide, Azide, Chlorate
Ferricyanide, Dichlorophenol
Indophenol, Tetrazolium salt
31. Prokaryotic NR
Assimilatory NR Respiratory NR
Prokaryotic NR
Synechococcus spp
Klebsiella oxytoca
Bacillus subtilis
NAP
NAR
32. Prokaryotic NR
Paracoccus Pantotrophus
NAS NAR NAP
2 Subunit 3 Subunit 2 Subunit
Cytoplasmic Cytoplasmic Periplasmic
• NR binds to bis-molybdopterin guanine dinucleotide cofactor at the active
site
• Differ in number and nature of cofactors
33. Synechococcus NR
NirA NrtABCD NarB
70 kDa polypeptide
Binds to bis-MGD and iron sulphur center
Electron donor – Fdred received from PS-I
Gene Cluster
35. INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI-110012
E-mail: ranjeetranjaniari@gmail.com; ranjeet.kumar@icar.gov.in; Cel No: +91-08368958133
Carbon and Nitrogen Metabolism
BIOCHEM-501
Ranjeet R. Kumar
Senior Scientist
Lecture - 4
36. Respiratory Nitrate Reductase
Respiratory NR
NAP NAR
• 2 Subunit enzyme
• 90 kDa catalytic subunit (NapA)
• Binds to bis-MGD and 4 [Fe-S]
cofactor
• 16 kDa electron transfer subunit
(NapB)
• Binds to 2 C-type heme unit
• Membrane bound
• 3 subunits
• Quinol dehydrogenase
• 140 kDa bis-MGD subunit
• 60 kDa electron transfer unit
(NarH) binds to 4[Fe-S] cluster
37. Regulation of NR activity
Werner Kaiser’s
NR Active
ATP-Mg
ADP-Mg
NR-Kinase (CDPK,
SNF-1 like)
P
14-3-3
PP2A
Pi
14-3-3
NR Inactive
38. NR-Regulation
• Leaves – Photosynthesis Increased – NR active – NR-
dephosphorylated
• Photosynthesis decreased – NR Phosphorylated – Serine 543 –
Created phosphopeptide motifs – Binds to one or more 14-3-3 proteins
- Inhibit NR activity
• NR active – dephosphorylation of serine – Type 2A protein
phosphatase
• Regulatory phosphorylation site is conserved Seryl residue in Hinge 1
• Ser 543 – Spinach// Ser534 – Arabidopsis
• Phosphorylation of serine created canonical structure 14-3-3 protein
binding site
• NR-Kinase – SNF-1, CDPK, CDPK6, CPK3
39. NR activity is post-translationally regulated
• Reduction of nitrate to nitrite – heavy drain on the
reductant NADH and NADPH.
• Rate of nitrate reduction should not exceed the supply of
reductant nor the carbohydrate waste
• Nitrite generated is hazardous – mutagenic – diazotized
amino group
• Tight regulation because NR acts as signalling molecule for
NO production
40. 14-3-3 protein
• First identified as abundant protein in Brain
• Named based on their mobility on ion exchange chromatography and 2-DE
•
• Unusually highly conserved protein family
• Central regulatory role in plant, fungal and mammalian cells
• Binds to phsophopeptide motif
• Modulate the activity of the enzyme
• Regulate sub-cellular location targets
• Acts as adaptor protein
• Regulator of NR activity.
41. Nitrite Reductase
NiR
Single peptide
Mol Wt. 61-64 kDa
Sedimentation coeff. 4.1s
Fe4-S4
Contain Siroheme
Reddish Brown
Chloroplastic e donor - Fd
Siroheme – Iron tetrahydroporphyrin of isobacteriochlorin type with 8
carboxylic acid containing side chain
42. Nitrite Reductase
• NiR – Transfer of 6e- for reduction of NO2- to NH4+
• 6e- transfer – Sulphite reductase
Siroheme
Fe4 – S4
NO2-
NO-siroheme
adduct
NH4+
Fd red
e-
1
e-
2
3
e-
4 e-
5 times
43. Nitrite Reductase Types
NiR
Multi Haem
Enzyme
Copper Containing
Enzyme
Cytochrome cd1 or
pseudomonas cyt oxidase
CcNiR – Cyt C NiR
NO
Variety of
products
45. INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI-110012
E-mail: ranjeetranjaniari@gmail.com; ranjeet.kumar@icar.gov.in; Cel No: +91-08368958133
Carbon and Nitrogen Metabolism
BIOCHEM-501
Ranjeet R. Kumar
Senior Scientist
Lecture - 5
46. Cyt CD1 or Pseudomonas cyt oxidase
• 4 Haem group with 2 polypeptide chains
• Catalyze reduction of NO2- to NO
• Tetra Haem enzyme
• 2 subunits
47. Cyt c Nitrite Reductase
• Multi Heme Enzyme
• Converts Nitrite to ammonia
• Fe binds to protoporphyrin IX ring
• Covalently linked to enzyme proteins
48. Copper based Nitrite Reductase
CuNIR
Plant Bacteria
• Presence of Type-I copper center in protein
• Type-I copper - bonded to 2 imidazole (Histidine) and thiolate
(cysteine)
• His bound to Type-II Cu centre – Responsible for binding and reducing
of NO2-
• Cys-His bridge : Facilitate rapid transfer of e- from type-I to type-II
centre
49. Glutamine Synthetase
Glutamate + ATP + NH3 ---------Glutamine + ADP + Phosphate + H2O
GS
GS-I
GS-II
GS-III
Eukaryotes
Decamer of identical
subunits
Prokaryotes
• Oligomer of 12
identical subunits
Bacteroides
Hexamer of
identical subunits
Largest in size
51. Ammonia Assimilation
NH4+
NO3- reduction
Glutamine
Symbiotic N2 fixation
Photorespiration
Phenylpropanoid
metabolism
Amino acid catabolism
• Pea, beans – 3 GS genes
• Cytosolic GS – Soybean induced by ammonia
• The regulation of GS is substrate specific
• NO3- supply leads to 2 fold increase in the GS expression
• Ammonia lower activity of GS by 40%
• Lemna minor – reversible inactivation of GS by darkness
52. Glutamine Synthetase
GS
Genes
Alpha
Gamma
40 kDa
40 kDa
Octameric
Phaseolus vulgaris
Beta
40 kDa
Gln-α
Gln-β
Gln-ϒ
• Protein structure – 12 identifical subunit
• Arranged in two layer of 6
• Active site has pair of Mn2+
53. Central role of GS in Plant N Metabolism
Glu ----------------------- Gln
NH3
GS
vegetative
reproductive
Tissue,
cell
and
sub-
cellular
distribution
Environmental
condition
Metabolic
status
Organ Distribution
AA
Amides
Transport
compounds
Proteins
NO3-
NH4+
N2
Sec. Metabolites
Photorespiration
57. Photorespiratory Nitrogen Cycle Involving
Respiratory Glutamate Metabolism
GDC – Glycine decarboxylase
GGAT – Glutamate:Glyoxalate amino transferase
SHMT – Serine hydroxy methyl transferase
SGAT – Serine glyoxalate amino transferase
CH2-THF – N5-N10- Methylene Tetra hydrofolate
• Amino acid are exported through pholem sap
• Glutamine was most abundant aa in pholem sap
• Major N-transporting form – Glutamine and Asparagine
• Most abundant aa in pholem sap – Gln, Asn, Ser, Pro