2. The activity of the enzyme is regulated by the reversible covalent modification of adenylation to a tyrosine residue on each of the 12 subunits.
3. The activity of GS is allosterically controlled by cumulative feedback inhibition by eight small molecules: tryptophan, histidine, carbamyl phosphate, glucosamine-6-phosphate, CTP, AMP, alanine, and glycine. The amide nitrogen of glutamine is directly involved in the biosyntheis of each of these compounds with exception of alanine and glcyine.
A single sensing mechanism is used to control the biosynthesis of GS and the covalent modification mentioned in 1 and 2; the global nitrogen regulatory (ntr) system. It is composed of four enzymes: a uridylyltransferase/uridylyl-removing enzyme (UTase/UR), encoded by the glnD gene, a small trimeric protein, PII, encoded by glnB, and a two-component regulatory system composed of the histidine protein kinase NtrB and the response regulator NtrC.
When cells are nitrogen limited, UTase covalently modifies PII by addition of a UMP group at a specific tyrosine residue, Try-51 on each subunit of the protein and the resultant uridylylated form of PII promotes deadenylylation of GS by ATase. Conversely, under nitrogen sufficient conditions, the uridylyl-removing activity of GlnD (Utase/UR) predominates and the deuridylylated PII promotes adenylylation of GS by ATase inactivating the enzyme. And so UTase/UR and PII, acting together, can provide a mechanism for sensing the intracellular nitrogen status where the Utase/UR protein controls the activites of ATase indirectly via PII.
In enterics, glnA is part of a complex operon, glnAntrBC, in which ntrB encodes a sensory histidine protein kinase, and ntrC a response regulator typical of bacterial two component sensing systems. The glnA gene is expressed from two tandem promoters glnAp1 and glnAp2, whereas the downstream ntrBC genes are expressed either by readthrough from the glnA promoters or from a separate promoter, pntrBC, situated between glnA and ntrBC.
Under nitrogen-sufficient conditions, glnA is expressed from glnAp1, which is transcribed by the major form of RNA polymerase, σ70. Most of these transcripts terminate at a Rho-independent terminator just downstream of glnA, and expression of ntrBC occurs primarily from pntrBC.
Under nitrogen-limiting conditions, the binding of NtrC to sites that overlap glnAp1 serves to repress expression from glnAp1 while activating transcription from glnAp2 by RNA polymerase containing the sigma factor σ54. σ54 or σN is an alternative sigma subunit of RNA polymerase that replaces, but is not homolgous to the standard σ70. It is used in the transcription of most genes involved in nitrogen metabolism and requires specific activator proteins that bind upstream of the protmoter and, through ATP hydrolysis, causes a conformation change in σ54 allowing it to undergo transition to the transcriptionally competent open promoter complex. This transcription is at a considerably elevated level comparde with that from glnAp1, and a proportion of these transcripts read through into ntrBC. The consequence of this organization is to provide a low level of glnA ntrBC transcription under conditions of nitrogen sufficiency, resulting in an elevation in transcription, approximately 14-fold, when fixed nitrogen is limiting.
NtrC (sometimes referred to as NRI) must be phosphorylated to activate transcription.
There are two known sources of phosphryl groups for the formation of NRI –P. 1.Phosphyrl groups can be directly transferred from the phosphrylated metabolic intermediate acetyl phosphate.
2. The autophosphorylated form of the glnL/ntrB product known as NRII. NRII binds ATP and becomes autophosphorylated on a histidine residue and on incubation with NRI, this phosporyl group is transferred to an aspartate residue, Asp-54, within the N-terminal domain of NRI.
The combination of NRII and the unmodified form of PII when nitrogen is in excess results in the very rapid dephosphorylation of NRI(NtrC)-P, stimulated by ATP. Hence down regulation of the operon.
NtrC-P is responsible for the regulation of genes responsible for nitrogen fixation (nif genes) as it relays that nitrogen levels are low. It also regulates the genes required for nitrate and nitrite assimilation (nasFEDCBA) and various other genes implicated in nitrogen regulation.
In the fixation process N2 is reduced to ammonium and the ammonium converted to organic form.
N2 + 3H2 3NH3
This reaction is thermodynamically favourable ΔG = -7.7 Kcal mol –1. However, the kinetic barrier to breaking the nitrogen – nitrogen triple bond that must be overcome, using a catalyst and/or the input of sufficient energy is high. The catalyst is the enzyme nitrogenase. Six electrons must be transferred by this enzyme to reduce N3 to 2NH3 and two ATPs must be hydrolysed per electron transferred. Hence nitrogen fixation is an energetically costly process and but be regulated so that if other forms of nitrogen are available these are utilized foremost.
Nitrogenase consists of two component proteins. The Fe protein which is a dimer of two identical subunits that symmetrically coordinate a single [4Fe-4S] cluster and a MoFe protein which is an α2β2 heterotetramer. It is composed of homolgous α and β subunits, that contain 2 copies each of the FeMo cofactor. The enzyme is very sensitive and so must be protected from inactivation by molecular oxygen as it is full of low potential redox centers. In fixation the Fe protein is reduced by either a ferrodoxin or flavodoxin electron donor. The reduced Fe protein hydrolyses 2ATP (only when in complex with MoFe) and undergoes a conformational change where the electron is transferred to the MoFe protein. The Fe protein then dissociates and the cycle is repeated.
Throughout the process eight electrons are actually consumed with two being lost as hydrogen. This is a wasteful reaction although many bacteria synthesise a membrane bound hyderogenase enzyme, which allows hydrogen to be used as an electron donor to the respiratory chain and thus recoup some of the metabolic energy expended in its production.
Regulation of nitrogen fixation is through as mentioned, regulation of the nif operon;
Q B A L F M Z W V S U X N E Y K D H J
Nif operon in klebsiella pneumoniae
The operons of the nif gene cluster are σ54 dependent. The activator of the nif gene cluster is NifA. The unmodified form of NifA is active.
NifH and NifD and NifK are structural proteins; the Fe protein and Mo protein α and β subunits respectively. NifQ,S,V,U,E,N,B,Z,W amd M are involved in the nitrogenase biosynthesis and NifF and NifJ in the electron transfer to nitrogenase. NifA nad NifL being the transcriptional regulators of the nif gene cluster; NifA the activator as mentioned and NifL a repressor.
As provision and operation of the nitrogenase system is very costly, transcription of the nif cluster is subject to tight control such that transcription occurs only in the absence of a fixed nitrogen source and transcription is repressed if oxygen concentrations are high enough to damage nitrogenase (determined by nifL).
The control of nif gene transcription is shown diagrammatically below;
NifA levels are under the control of the NtrBC system. Where NtrC-phosphate binds as a transcriptional activator. The glnA, nas and nifA promoters have decreasing affinities for NtrC-phosphate resulting in a hierarchy in the utilization of nitrogen sources with high levels of ammonia being used in preference to low levels of ammonia which in turn is favoured over nitrate which is in turn used in preference to nitrogen fixation.
NifL is an FAD-containing redox sensor which when oxidized binds to NifA and prevents NifA from activating nif gene transcription. In the reduced state NifL is sequestered at the membrane. The oxidation state of nifL is controlled by the activity of the respiratory electron transport chain in the membrane so that when it can no longer keep NifL reduced it is released from the membrane.
During symbiotic fixation by bacteroids in the root nodule regulation by the NtrBC system is overridden so regulation is only determined by oxygen tension and not by nitrogen availability as plants utilize the excess nitrogen produces.
The oxygen concentration of the root nodules is kept very low and so the peribacteroid space contains high concentrations of the protein leghaemoglobin, a heam containing oxygen binding protein. It is used to store and scavenge oxygen.
The fix genes are thus required in the symbiotic state, which encode for the production of heam for leghaemooglobin and also for a cytochromoe c oxidase which has exceedingly high affinity for oxygen. FixLJ is the two component regulatory system whereby heam in a globin like domain of FixL reversibly binds oxygen. The deoxy form of fixL acts as a Fix J kinase and thus signals the transcription of the fix operon. The oxy form acts as a FixJ phosphatase.
Most bacteria can also use nitrate as a nitrogen source. The nitrate and nitrite reductase involved in this process are cytoplasmic enzymes, distinct from those participating in the respiratory process of denitrification.
NO3- + 2H+ + 2e- NO2 + H2O nitrate reductase
NO2- + 8H+ + 6e- NH4+ + 2H2O nitrite reductase
Expression of nas operon encoding these enzymes is also NtrC/σ54 regulated as mentioned. nas is also nitrate and nitrite regulated. Nitrate is not used as a source when ammonium levels are high saving the cell providing low potential reductants or additional enzymes. In such bacteriods sym plasmids carry the nif, nod and fix genes (where nod genes are required for the nodule production and maintenance.)
Many cyanobacteria are capable of nitrogen fixation, and in some cases this occurs in highly developed cells termed heterocysts that differentiate from the vegetative cell filaments. Both nitrogen fixation and heterocyst development are also subject to ammonium repression.
There is evidence for ntr analogous systems in bacreria other than those mentioned. There is however, little evidence of a classical ntr system in grampositive bacteria, nor is there any suggestion of an alternative global regulatory system that characterizes this group.
To summerise nitrogen metabolism is imperative for life providing the necessary constituents of living tissue. It is highly regulated to prevent utilization of excess energy, through various systems, with the ntr system being the most common.
