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Assimilation of ammonia

Assimilation of ammonia. glutamine synthetase (GS). Glutamine synthetase of Salmonella thyphymurium (a bacterium closely related to E. coli ). Ciclo del nitrógeno. fig 22.1 Lehninger. fig 22.2 Lehninger.

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Assimilation of ammonia

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  1. Assimilation of ammonia glutamine synthetase (GS) Glutamine synthetase of Salmonella thyphymurium (a bacterium closely related to E. coli)

  2. Ciclo del nitrógeno fig 22.1 Lehninger

  3. fig 22.2 Lehninger

  4. All nitrogenases have an iron- and sulfur-containing cofactor that includes heterometal atom in the active site (e.g. FeMoCo). In most, this heterometal is molybdenum, though in some species it is replaced by vanadium or iron. fig 22.3 Lehninger

  5. Lehninger Figure 22-04a

  6. Reaction in assimilation of ammonia and major fates of the nitrogen atoms

  7. Glucose (outside) Fructose - - Glucose-6-P (inside) Fructose-1-P Fructose-6-P - + - Fructose-1,6-diP DHAP Glyceraldehyde-3-P Glycerate-1,3-diP Glycerate-3-P Inducer Exclusion Glycerate-2-P glucose (outside) IIAglc PEP IIB-IIC~P EI Hpr~P - + Hpr IIAglc~P EI~P IIB-IIC Pyruvate PDH glucose-6-P (inside) CO2 CO2 Activate AC acetyl~CoA acetyl~P Oxaloacetate acetate Citrate Malate Isocitrate acetyl~CoA NH3 aspartate CO2 + + ICD + Glyoxylate GDH GOGAT Fumarate 2-ketoglutarate Glutamate ODH GS NH3 CO2 Succinate Succinyl~CoA Glutamine Ammonia assimilation is tied to the flux of carbon through central metabolic pathways The 2-ketoglutarate/glutamine ratio is a signal of the cellular nitrogen status

  8. Some of the reactions involving glutamine

  9. Glutamate dehydrogenase (GDH) In bacteria the Km for ammonium is high (~ 1mM), thus the enzyme cannot contribute to ammonia assimilation when ammonia is limiting. In mammals, the enzyme is mitochondrial and participates in ammonia excretion. Yeast have 2 enzymes, an NADPH enzyme forms glutamate and an NADH enzyme forms a-ketoglutarate.

  10. The glutamate synthase (GOGAT) reaction

  11. Under conditions of ammonia limitation, the GS-GOGAT cycle is used for ammonia assimilation in bacteria and plants 2-ketoglutarate + NH3 + NAD(P)H + H+ glutamate + NAD(P)+ GDH glutamate + ATP + NH3 glutamine + ADP + Pi GS glutamine + 2-ketoglutarate + NADPH + H+ 2 glutamate + NADP+ glutamate synthase (GOGAT) Sum of GS + GOGAT: 2-ketoglutarate + NH3 + ATP + NADPH + H+ glutamate + ADP + Pi + NADP+

  12. Salmonella thyphimurium GS top view showing ADP and 2 Mn Adjacent subunits form the active sites

  13. Glutamine synthetase reaction mechanism ATP binds to GS glutamate binds to (E.ATP) E.ATP.glu ----> E.ADP.glutamyl-g-P conformational change favors NH4+ binding deprotonation of NH4+ by an Asp causes a flap (324-328) to close over active site ammonia attacks glutamyl-g-P forming tetrahedral intermediate Pi and a proton are lost The flap opens and glutamine leaves

  14. Regulation of E. coli glutamine synthetase E. coli is reported to be regulated in three distinct ways: 1. Cumulative feedback inhibition 2. Reversible covalent modification (adenylylation) 3. Regulation of enzyme synthesis

  15. Cumulative feedback inhibition of GS The enzyme is inhibited by the following compounds: alanine, glycine, tryptophan, histidine, carbamyl phosphate, glucosamine-6-phosphate, CTP, and AMP Each of the inhibitors provides only partial inhibition, complete inhibition requires all of the inhibitors. Kinetic studies suggested that none of the inhibitors was competitive with substrates. BUT-Structural studies show a different picture: AMP binds at the ATP substrate site Gly, ala, and ser bind at the glu site carbamyl phosphate binds overlapping the glu and Pi sites the binding of carbamyl phosphate prevents the binding of ala, gly, and ser.

  16. GS is regulated by reversible covalent adenylylation ATase ATP PPi GS~AMP GS (inactive) (active) ADP Pi ATase

  17. The activity and level of Glutamine Synthetase (GS) are regulated by the ratio of carbon to nitrogen add glucose to 1% add glutamine to 0.2% Nutrient broth culture (N>C) C>N N>C level of GS is low level of GS is high level of GS is low GS mostly adenylylated GS mostly unadenylylated GS mostly adenylylated

  18. GS is regulated at both the transcriptional and post-transcriptional levels Ammonia scarce Ammonia plentiful GS not adenylylated GS adenylylated glnA gene highly expressed glnA gene not highly expressed A large amount of very A small amount of enzyme that active enzyme is mostly inactive

  19. GS PII GS-AMP PII-UMP UTase/UR/PII monocycle UTase/UR/PII monocycle Two bicyclic cascades control GS synthesis and activity a-ketoglutarate NRII PII ATP NRI~P NRII gln gln UR UTase/UR UT PII-UMP ADP NRI NRII~P a-ketoglutarate gln gln AR ATase AT gln gln UR UTase/UR UT ATase/GS monocycle

  20. KNTase 13-RMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTDGPYSDIEMMCVMSTEE-(2)-FSHEWIT * * * **** ** * **** DNA POLb 154-MLQMQDIVLNEVKKL-DPEY-IATVCGSFRRGAES-SGDMDVLLTHPNFT-(31)-TKFMGVC * * * **** ** * **** E. c. UTase/UR 68-IDQLLQRLWIEAGFSQIADL-ALVAVGGYGRGELHPLSDVDLLILSRKKL-(6)-KVGELLT AA A N N Figure 10. Alignment of the known active sites from kanamycin nucleotidyl transferase and rat DNA polymerase b with theN-terminal part of the UTase/UR. The structures of KNTase and Polb are known. Below the UTase/UR sequence, the locations of the G93A, G94A, G98A, D105N, and D107N mutations in glnD are shown. glutamine UTase PII PII~UMP (N-rich) (N-poor) UR glutamine Uridylyltransferase/uridylyl-removing enzyme measures glutamine and controls the activity of PII

  21. E. coli PII (top view) E. coli PII (side view) T-Loop C-Loop B-Loop E. coli PII subunit Cyanobacterial PII (top view) Structure of the unliganded form of PII

  22. ATase + PII GS + ATP GS~AMP + PPi gln Biphasic response of GS adenylylation reaction to 2-KG

  23. PII contains non-equivalent 2-KG binding sites Binding of 2-KG to PII (30 mM) when ATP is present in excess

  24. No interaction with ATase or NRII Kd~ 5 mM 2-ketoglutarate low Gln UMP Interacts with ATase and NRII high Gln high Gln low Gln UMP UMP Kd~ 150 mM UMP low Gln No interaction with ATase or NRII No interaction with ATase of NRII high Gln PII protein integrates antagonistic signals [Uridylylation reduces negative cooperativity in 2KG binding]

  25. glutamine uridylyl group a-ketoglutarate PII protein integrates antagonistic signals NRII (NRI kinase) NRII::PII (NRI~P phosphatase) ATase ATase::PII (AT activity) ATase::PII-UMP (AR activity) UTase/UR (UT activity) PII PII-UMP UTase/UR (UR activity) a-ketoglutarate glutamine

  26. Reconstitution of the UTase/UR-PII monocycle At physiological concentration of 2-KG and gln, only gln regulates PII uridylylation state.

  27. Reconstitution of the UTase/UR-PII-ATase-GS bicycle both gln and 2KG regulate the bicycle Only gln regulates the UTase/UR-PII monocycle

  28. Glutamine regulates the phosphorylation state of NRI by acting on UTase/UR

  29. Response of the bicyclic system to glutamine addition

  30. 2-Ketoglutarate regulates NRI phosphorylation state, but not PII uridylylation state in the bicyclic system

  31. The two bicycles respond differently to glutamine

  32. “Level 1” glnALG GS NRI NRII PII NRII NRI~P Signals nac Ntr genes nifLA glnK “Level 2” GlnK Nac NifA NifL activation and repression of genes nif genes “Level 3” Gene cascade controlling nitrogen assimilation and fixation

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