Converting N2 into a Biologically Useful Form.  Nitrogen is the third most abundant element on earth.  The conversion of N2 into its many chemical forms is described by the Nitrogen Cycle (Figure 1).  Microscopic viruses, plants and animals all require nitrogen as an essential component of life.  Over 70% of the earth’s atmosphere is Nitrogen, O2 and CO2 comprise the rest.  Cells and living organisms can use nitrogen, only after it is reduced to its primary biologically usable form, ammonia.  Reduction of N2 is carried out by a special group of organisms living in the soil that are the only organisms able to convert atmospheric N2 into a biologically usable form.  These are the nitrogen fixing bacteria which convert the atmospheric nitrogen into ammonia, and they are essential to the persistence of life on this planet.

Biological Assimilation of Nitrogen Compounds.  After nitrogen is reduced to ammonia, it can be used by organisms for conversion to other biological molecules.  Ammonia can be oxidized to nitrite and ultimately nitrate by soil bacteria, which derive their energy from these conversion processes that are called nitrification.  Many plants and bacteria have a class of enzymes called reductases that can absorb soil nitrate and reduce it back to ammonia (Figure 1).  In plants ammonia is a source of nitrogen to make essential components of life including amino acids and nucleic acids.  Animals that ingest plants use these amino acids as building blocks for protein, and other nitrogen containing compounds (Nelson and Cox, 2004).

Amt Family of Membrane Proteins.   Biosynthesis of essential molecules requires reduced nitrogen.  In some eukaryotes this nitrogen is obtained as amino acids or nitrates, however in most prokaryotes the source of biological nitrogen is ammonia or ammonium.  Diffusion of ammonia across cellular plasma membranes under acidic conditions tends to be slow and can prohibit the cell from maintaining homeostasis.  Therefore, organisms have developed intricate systems to control the uptake of ammonia using a family of membrane proteins, Amt, found in bacteria, archae, fungi, and plants.

High-resolution crystal structures of Amt proteins reveal that they exist as trimers with 11 membrane-spanning helices in each monomer.  The combination of the monomers forms a bundle around a hydrophobic channel (Khademi et al., 2004).  This structure suggests that Amt proteins act as channels across which uptake of ammonia by a cell occurs (Figure 2).  The Amt channel, which does not actively transport ammonium, cooperates with other ammonia transporters to regulate the amount of ammonia in the cell.  Each monomer of Amt has an ammonium-binding site on the extracellular surface which deprotonates the ammonium ion to ammonia before it passes through the channel.  Once inside the cell the ammonia can be protonated to ammonium.  Two important features of Amt channels are that they do not require energy to transport ammonia across the plasma membrane, as many active transport channels do, and that ammonia transport across this channel can be easily regulated by PII proteins.

PII Proteins.  Nitrogen metabolism in archae and bacteria is regulated by soluble PII proteins.  PII proteins have recently been discovered in eukaryotes, and the structure has been determined in plants, as described in Wendy Ingram’s Biochemistry 462b Honors project Plant PII Protein Structure: Insights into Eukaryotic Nitrogen Metabolism (2007).   These highly conserved proteins are among the most ancient, and versatile signaling proteins.  PII proteins are small trimeric signal transduction proteins that regulate gene transcription, modulate activity of regulatory proteins, and control the catalytic activity of nitrogen metabolism.  Two PII proteins in E.coli are GlnB and GlnK and they can bind ATP, 2-ketoglutarate, and magnesium (Van Heeswijk et al., 1996).  A great deal is still not known about these specific proteins but they form 1:1 complexes with Amt channel proteins.  This Amt/GlnK complex tends to dissociate in the presence of ATP, magnesium chloride, and 2-ketoglutarate.  By studying the structure of GlnK1, Yildiz et al. (2007) gained insight into the structure and function of the Amt/GlnK complex.