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

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.

Lehninger Figure 22-04a

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

Glucose (outside)

Glucose-6-P (inside)

Fructose-6-P

Fructose-1,6-diP

PEP

Pyruvate

acetyl~CoA

Glyceraldehyde-3-P

Glycerate-1,3-diP

Glycerate-3-P

Glycerate-2-P

DHAP

acetyl~P

acetate

EI

Hpr

IIAglc

IIB-IIC

glucose(outside)

glucose-6-P(inside)

EI~P

Hpr~P

IIAglc~P

IIB-IIC~P

Oxaloacetate

Malate

Succinate

Citrate

Isocitrate

2-ketoglutarate

Succinyl~CoA

Glyoxylate

Fumarate Glutamate

Glutamine

NH3

NH3acetyl~CoA

aspartate

CO2

CO2

CO2CO2

Inducer Exclusion

Activate AC

Fructose

Fructose-1-P

-

-

-

-

-

+++

+

+

ODH

PDH

GDHGOGAT

GS

ICD

Ammonia assimilation is tied tothe flux of carbon through centralmetabolic pathways

The 2-ketoglutarate/glutamine ratiois a signal of the cellular nitrogen status

Some of the reactions involving glutamine

Glutamate dehydrogenase (GDH)

In bacteria the Km for ammonium is high (~ 1mM), thus the enzyme cannot contribute toammonia assimilation when ammonia is limiting. In mammals, the enzyme is mitochondrialand participates in ammonia excretion. Yeast have 2 enzymes, an NADPH enzyme formsglutamate and an NADH enzyme forms -ketoglutarate.

The glutamate synthase (GOGAT) reaction

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+

Salmonella thyphimurium GS

top view showing ADP and 2 Mn

Adjacent subunits form the active sites

Glutamine synthetase reaction mechanism

ATP binds to GS

glutamate binds to (E.ATP)

E.ATP.glu ----> E.ADP.glutamyl--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--P forming tetrahedral intermediate

Pi and a proton are lost

The flap opens and glutamine leaves

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

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, completeinhibition requires all of the inhibitors.

Kinetic studies suggested that none of the inhibitors was competitivewith substrates.

BUT-Structural studies show a different picture:

AMP binds at the ATP substrate siteGly, ala, and ser bind at the glu sitecarbamyl phosphate binds overlapping the glu and Pi sitesthe binding of carbamyl phosphate prevents the binding of ala, gly, and ser.

GS is regulated by reversible covalent adenylylation

GS(active)

GS~AMP(inactive)

ATase

ATase

ATP PPi

ADP Pi

The activity and level of Glutamine Synthetase (GS) are regulated by theratio 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 glucoseto 1%

add glutamineto 0.2%

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 thatactive enzyme is mostly inactive

Two bicyclic cascades control GS synthesis and activity

PII

PII-UMP

GS

GS-AMP

UTase/UR UTURgln gln glngln

ATaseAR AT

ketoglutarate

UTase/UR/PII monocycle ATase/GS monocycle

PII

PII-UMP

UTase/UR UTURgln gln

NRI~P

NRI

NRIIATP

ADP

ketoglutarate

NRII~P

NRII

UTase/UR/PII monocycle

Uridylyltransferase/uridylyl-removing enzyme measures glutamineand controls the activity of PII

PII PII~UMPUTase

UR

glutamine

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.

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

Biphasic response of GS adenylylation reaction to 2-KG

GS + ATP GS~AMP + PPi gln

ATase + PII

Binding of 2-KG to PII (30 M) when ATP is present in excess

PII contains non-equivalent 2-KG binding sites

[Uridylylation reduces negativecooperativity in 2KGbinding]

Kd~ 5 M

Kd~ 150 M

No interaction withATase or NRII

Interacts with ATaseand NRII

No interaction withATase or NRII

UMP

UMPUMP

UM

P

2-ketoglutarate

low Gln

high Gln

low Gln

high Gln

No interaction withATase of NRII

high Gln low Gln

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)

UTase/UR(UR activity)

glutamine

-ketoglutarate

uridylyl group

PII

PII-UMP

-k

eto

glu

tara

te

glutamine

PII protein integrates antagonistic signals

Reconstitution of the UTase/UR-PII monocycle

At physiological concentration of 2-KG and gln, only gln regulatesPII uridylylation state.

Reconstitution of the UTase/UR-PII-ATase-GS bicycle

both gln and 2KGregulate the bicycle

Only gln regulatesthe UTase/UR-PIImonocycle

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

Response of the bicyclic system to glutamine addition

2-Ketoglutarate regulates NRI phosphorylation state, but not PIIuridylylation state in the bicyclic system

The two bicycles respond differently to glutamine

Gene cascade controlling nitrogen assimilation and fixation

glnALG

GS NRI NRII

NRI~P

NRIIPII

glnK

GlnK

nifLA

NifA NifL

nif genes

Ntr genes

Signals

“Level 1”

“Level 2”

“Level 3”

nac

activation andrepression of genes

Nac