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- 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
- 18. Properties of NT/ NiT from Eukaryotes
- 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
- 20. Nitrate/ Nitrite Transport System in Chlamydomonas Nitrate assimilatory genes cluster 45 Kb 15 Kb • Nia-1 – Nitrate reductase • Nii-1 – Nitrite reductase • Nrt-2 – Nitrate/nitrite transporter • Nrt-2 – Transporter • Aox-1 – Alternate oxidase
- 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
- 50. Glutamine Synthetase Plant GS-I GS-II Cytosol Chloroplast Universal • Oldest existing enzyme • Catalyse assimilation of ammonia to form glutamine
- 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
- 54. GS-GOGAT Cycle GS GOGAT Glutamine Glutamate ATP ADP + Pi NH4+ α-Ketoglutarate Glutamate NAD(P)H or 2Fdx- NAD(P)+ or 2Fdx Azaserine Methionine Sulfoximine L-Phosphinothricin
- 55. GOGAT • GOGAT – Glutamine: 2-oxyoglutarate amido transferase • Fd-GOGAT – Cyanobacteria and photosynthetic Eukaryotes • NADPH - GOGAT – Non-photosynthetic bacteria and Archae • NADH - GOGAT – Fungi, Diatoms
- 56. Photorespiratory Nitrogen Cycle Involving Respiratory Glutamate Metabolism Chloroplast Peroxisome Mitochondria 2-oxoglutarate GDH Glutamate Glycine Serine CH2-THF NH4+ SHMT 2-oxoglutarate Glycine Serine GDC CO2 2e- NH4+ 2-oxoglutarate Glutamate Glycolate Glyoxylate GGAT OH-Pyruvate Glycolate Glycerate Glutamate SGAT Glutamate Glutamine GOGAT GS2 RuBP G3P NH4+ Glutamine GS1 NH4+
- 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