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. fig 22.3 Lehninger 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.

5. Lehninger Figure 22-04a

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

7. 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  -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 + NH 3 + NAD(P)H + H + glutamate + NAD(P) + GDH glutamate + ATP + NH 3 glutamine + ADP + Pi GS glutamine + 2-ketoglutarate + NADPH + H + 2 glutamate + NADP + glutamate synthase (GOGAT) Sum of GS + GOGAT: 2-ketoglutarate + NH 3 + 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-  -P conformational change favors NH 4 + binding deprotonation of NH 4 + by an Asp causes a flap (324-328) to close over active site ammonia attacks glutamyl-  -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 GS (active) GS~AMP (inactive) ATase ATP PPi ADP Pi

17. The activity and level of Glutamine Synthetase (GS) are regulated by the ratio of carbon to nitrogen 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 add glucose to 1% add glutamine to 0.2%

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

19. Two bicyclic cascades control GS synthesis and activity PII PII-UMP GS GS-AMP UTase/URUTUR gln ATaseARAT  ketoglutarate UTase/UR/PII monocycle ATase/GS monocycle PII PII-UMP UTase/URUTUR gln NRI~P NRI NRII ATP ADP  ketoglutarate NRII~P NRII UTase/UR/PII monocycle

20. Uridylyltransferase/uridylyl-removing enzyme measures glutamine and controls the activity of PII PII PII~UMP UTase UR glutamine (N-rich)(N-poor) KNTase 13-RMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTDGPYSDIEMMCVMSTEE-(2)-FSHEWIT * * * **** ** * **** DNA POL  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  with theN-terminal part of the UTase/UR. The structures of KNTase and Pol  are known. Below the UTase/UR sequence, the locations of the G93A, G94A, G98A, D105N, and D107N mutations in glnD are shown.

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

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

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

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

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

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