Nitrogen assimilation/the GS pathway Flashcards
bacteria acquire nitrogen from a variety of sources
N2, NO3-, NO2-, NH3, amino acids and urea
whichever source is used, N is converted to ammonia then assimilated
when ammonia is assimilated, it produces the amino acids
glutamate and glutamine
glutamate and glutamine are amino acid group donors in the biosynthesis of
other amino acids
nitrogenous DNA bases (purine and pyrimidine)
NAD+
other N containing molecules (PABA, para-aminobenzoic acid growth factor)
Ammonia is assimilated by 2 enzymes
GDH glutamate dehydrogenase (high N, does not use ATP)
GS glutamine synthetase (low N, uses ATP)
GDH reaction
ammonia > glutamATE
NH3 + 2oxo-glutarate + NADPH + H+»_space; GLUTAMATE + NADPH
GS reaction
ammonia > glutamINE
NH3 + glutamate + ATP»_space; GLUTAMINE + ADP + Pi
GOGAT
glutamine-oxoglutarate aminotransferase
manufactures glutamate from glutamine and α-ketoglutarate, and thus along with glutamine synthetase
2-oxoglutarate + NADPH + H+ + glutamine»_space; 2 glutamate + NADP+
GS+GOGAT is equivalent to GDH pathway but requires ATP (done only in low N concs)
GS pathway summary
NH4+ + 2oxoglutarate + NADPH + ATP»_space; glutamate + NADP+ + ADP + Pi
the first reaction makes a glutamine product (GS)
the second reaction (GOGAT) makes the glutamate product by reacting with 2 oxoglutarate
Forms the same product as GDH but requires ATP so is less favoured and only functions when cellular N is low
GS is regulated at 3 levels:
allosterically
post translationally
transcriptionally
Allosteric regulation of GS involves
cumulative feedback inhibition
various N containing compounds bind to GS to reduce its activity (allostery) e.g. NAD
together they can reduce GS activity by 90%
this allows GS activity to be adjusted to the current demand of N from a range of different pathways
Post translational regulation of GS involves
adenylylation (AMP) on tyrosine
adenylyl transferase adds the AMP. Makes GS LESS active and MORE susceptible to cumulative feedback inhibition
The structure of GS
12 subunits in 2 rings each with a specific tyrosine residue that can be adenylylated
so it can hold 12 AMPs which reduce activity
How does the cell measure the nitrogen concentration?
The Gln:2-OG ratio
the glutamine to 2-oxoglutarate ration
The Gln:2-OG ratio
indicates the N status of the cell
both glutamine and 2 oxoglut have the same carbon skeleton but glutamine has 2N
when cellular N is LOW, the glutamine:2oxoglut ratio FALLS = LESS glutamine
when cellular N is HIGH, the glutamine:2oxoglut ratio RISES = MORE glutamine
both are measured to ensure it is just a N shortage, not a carbon one!
Which protein senses the Gln:2-OG ratio?
PII
UTase (uridylyl transferase) adds and removes a UMP to PII depending on the N concentration of the cell
when N is high = PII = GS-AMP = OFF
when N is low = PII-UMP = GS = ON
PII-UMP turns GS
ON
PII turns GS
OFF
Ultimately, GS is controlled by
PII and UTase
UTase is inhibited by
glutamine and activated by 2oxoglutarate (which is why when cellular N is LOW, the glutamine:2oxoglut ratio FALLS = LESS glutamine MORE oxoglutarate
UTase is inhibited by
glutamine
and activated by 2oxoglutarate (which is why when cellular N is LOW, the glutamine:2oxoglut ratio FALLS = LESS glutamine MORE oxoglutarate
PII-UMP turns GS on by
activating AT (adenylyl transferase) to remove AMP from GS
When cellular N is low, PII is
uridylylated
PII-UMP
Removing AMP from GS
activates GS
occurs when N is low
When cellular N is high
UR removes UMP from PII
AT puts AMP on GS
to inactivate GS
When cellular N is low
UTase puts UMP on PII
AT removes AMP from GS
to activate GS
PII is involved in both
transcriptional and post translational control of GS
