Lecture 2: Assimilation of ammonium Flashcards
How is ammonium assimilated into amino acids?
Ammonium is assimilated by being incorporated into glutamate and glutamine.
• Glutamate is the initial amino donor for the biosynthesis of around 90% of N-containing molecules.
• Glutamine is the amino donor for the rest.
How is the pathway regulated through σ54?
σ54 is part of the bacterial RNA polymerase.
• It recognises promoters with consensus sequences at positions -24 and -12 relative to the transcription start site.
• EBPs (enhancer binding proteins) are required for initiation.
• Productive interactions between the DNA-bound EBP and σ54 require DNA looping. This may be assisted by DNA-bending proteins such as integration host factor (IHF).
• Important EBPs include NtrC (most bacteria) and NifA (nitrogen fixers).
How does is ammonium sensed?
The NtrBC system controls expression of glutamine synthetase.
• We only want to use it when ammonium levels are low because the glutamate dehydrogenase reaction is energetically costly.
• The Ntr system senses nitrogen availability through the levels of glutamine in the cytoplasm.
• Glutamine is sensed instead of ammonium because the bacterium needs to know if the pool of N donors for metabolism is sufficient.
• The cell needs to be able to switch off the pathway once the N donor pool has been restored.
• The Ntr system also responds to cytoplasmic α-KG levels. Its concentration varies inversely with that of glutamine. α-KG signals the carbon status of the cell to the Ntr system.
• During the transition to nitrogen limitation, the glutamine pool drops up to 10-fold and the α-KG levels rise by an equivalent amount.
• If ammonium concentration drops, the glutamate DH pathway cannot operate. Any glutamine present is used to synthesise glutamine through the reverse glutamate synthase pathway. α-KG rises because it isn’t being used by glutamate synthase.
• Glutamine synthetase must be induced for ammonium assimilation.
What are PII proteins?
PII proteins are ubiquitous trimeric proteins found in bacteria.
• Many proteobacteria contain at least 2 PII homologues, often designated GlnB and GlnK., that are subject to modification by uridylyltransferase/uridylyl-removing enzyme (UT/UR).
• UT/UR is a bifunctional enzyme encoded by glnD.
• When intracellular of glutamine is relatively low, UT/UR uridylylates the PII ¬proteins.
• Conversely, when the concentration of glutamine is relatively high, the UT/UR favour the de-uridylation activity.
• The interaction of the PII signal proteins with their targets is also modulated by the carbon signal α-KG and by the ATP/ADP ratio.
• PII upregulates NtrB, which in turn upregulates NtrC.
• PII has binding sites for adenosine nucleotides and 2oxyglutarate (i.e. α-KG).
• 2-OG binding affects the PII T-loop structure, thereby altering the affinity between PII and its binding protein targets.
• PII can also physically block the ammonium transporter. Ammonium uptake is an active process. The ammonium transporter can be blocked in order to save energy and avoid ammonium toxicity.
• GlnK (PII) and AmtB (ammonia channel) form a complex. A loop of PII enters and blocks the channel.
How can glutamine synthase be post-transcriptionally regulated?
Glutamine synthetase can be post-transcriptionally regulated by AR and AT.
• AT/AR (adenylyl transferase/adenylyl removing enzyme).
• In high glutamine conditions, P¬II is activate. It upregulates the AT activity and causes GS to have an AMP group added. This inactivates it.
• In low glutamine conditions, P¬II is uridylylated. This activates the AR activity and causes the AMP group to be removed. GS is activated.
How does nitrate assimilation work?
Nitrate is the preferred source of nitrogen for plants.
• Many bacteria can also use nitrate as a nitrogen source.
• The assimilatory nitrate and nitrite reductases involved in this process are cytoplasmic enzymes.
• They are completely distinct from the nitrate and nitrite reductases participating in the respiratory process of denitrification.
• it takes twice as many electrons to reduce a nitrogen atom in nitrate to ammonium as it does to reduce a nitrogen atom in dinitrogen to ammonium.
• Expression of the assimilatory nitrate reductase/assimilatory nitrite reductase nas operon is also regulated by both NtrC and σ54.
• Nitrate is not used as nitrogen source when ammonium levels are high. This strategy saves the cell having to provide low potential reductant or produce additional enzymes.
• Nas is also nitrate and nitrite regulated.
