Transcript No Slide Title

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
All nitrogenases have an iron- and sulfurcontaining 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
Lehninger Figure 22-04a
Reaction in assimilation of ammonia and major fates of the nitrogen atoms
Glucose (outside)
Fructose
Glucose-6-P (inside)
-
-
Fructose-1-P
Fructose-6-P
-
+
-
Fructose-1,6-diP
DHAP
Ammonia assimilation is tied to
the flux of carbon through central
metabolic pathways
Glyceraldehyde-3-P
The 2-ketoglutarate/glutamine ratio
is a signal of the cellular nitrogen status
Glycerate-1,3-diP
Glycerate-3-P
Inducer Exclusion
Glycerate-2-P
PEP
+
CO2
Pyruvate
PDH
acetyl~CoA
aspartate
EI
Hpr~P
EI~P
Hpr
CO2
Oxaloacetate
Malate
glucose
(outside)
acetyl~P
+
Fumarate
Glyoxylate
Isocitrate
+
IIB-IIC~P
IIAglc~P
IIB-IIC
Activate AC
ICD
+
CO2
NH3
GDH
GOGAT
2-ketoglutarate
ODH
Glutamate
GS
CO2
Succinate
Succinyl~CoA
glucose-6-P
(inside)
acetate
Citrate
acetyl~CoA
IIAglc
Glutamine
NH3
Some of the reactions involving glutamine
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.
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 + 2-ketoglutarate + NADPH + H+
glutamine + ADP + Pi
GS
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-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
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, 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.
GS is regulated by reversible covalent adenylylation
ATase
ATP
GS
(active)
PPi
ADP
Pi
ATase
GS~AMP
(inactive)
The activity and level of Glutamine Synthetase (GS) are regulated by the
ratio of carbon to nitrogen
add glucose
to 1%
Nutrient broth culture
(N>C)
level of GS is low
GS mostly adenylylated
add glutamine
to 0.2%
C>N
level of GS is high
GS mostly unadenylylated
N>C
level of GS is low
GS mostly adenylylated
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
active enzyme
A small amount of enzyme that
is mostly inactive
Two bicyclic cascades control GS synthesis and activity
a-ketoglutarate
ATP
NRII
NRI~P
NRII
PII
gln
ADP
PII-UMP
UTase/UR/PII monocycle
GS
PII
UR
UTase/UR
UTase/UR UT
NRI
NRII~P
a-ketoglutarate
gln
UR
UT
gln
gln
AR
ATase
AT
PII-UMP
GS-AMP
UTase/UR/PII monocycle
ATase/GS monocycle
gln
gln
Uridylyltransferase/uridylyl-removing enzyme measures glutamine
and controls the activity of PII
glutamine
UTase
PII
(N-rich)
UR
PII~UMP
(N-poor)
glutamine
KNTase
DNA POLb
E. c. UTase/UR
13-RMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTDGPYSDIEMMCVMSTEE-(2)-FSHEWIT
*
*
*
**** **
* ****
154-MLQMQDIVLNEVKKL-DPEY-IATVCGSFRRGAES-SGDMDVLLTHPNFT-(31)-TKFMGVC
*
*
*
**** **
* ****
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.
Structure of the unliganded form of PII
E. coli PII (top view)
E. coli PII (side view)
T-Loop
C-Loop
B-Loop
E. coli PII subunit
Cyanobacterial PII (top view)
Biphasic response of GS adenylylation reaction to 2-KG
ATase + PII
GS + ATP
GS~AMP + PPi
gln
PII contains non-equivalent 2-KG binding sites
Binding of 2-KG to PII (30 mM) when ATP is present in excess
PII protein integrates antagonistic signals
No interaction with
ATase or NRII
Kd~ 5 mM
2-ketoglutarate
[Uridylylation
reduces negative
cooperativity in 2KG
binding]
low Gln
Interacts with ATase
and NRII
high Gln
Kd~ 150 mM
high Gln
No interaction with low Gln
ATase or NRII
high Gln
low Gln
No interaction with
ATase of NRII
PII protein integrates antagonistic signals
NRII::PII
(NRI~P phosphatase)
ATase
ATase::PII
(AT activity)
ATase::PII-UMP
(AR activity)
UTase/UR
(UT activity)
PII
PII-UMP
a-ketoglutarate
NRII
(NRI kinase)
UTase/UR
(UR activity)
glutamine
uridylyl group
a-ketoglutarate
glutamine
Reconstitution of the UTase/UR-PII monocycle
At physiological concentration of 2-KG and gln, only gln regulates
PII uridylylation state.
Reconstitution of the UTase/UR-PII-ATase-GS bicycle
both gln and 2KG
regulate the bicycle
Only gln regulates
the UTase/UR-PII
monocycle
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 PII
uridylylation state in the bicyclic system
The two bicycles respond differently to glutamine
Gene cascade controlling nitrogen assimilation and fixation
“Level 1”
glnALG
GS
NRI
NRII
PII
NRII
NRI~P
nac
Ntr genes
nifLA
Signals
glnK
“Level 2”
GlnK
Nac
activation and
repression of
genes
NifA
nif genes
NifL
“Level 3”