The first structure of an ammonia channel from the Amt/MEP/Rh protein superfamily, determined to 1.35 angstrom resolution, shows it to be a channel that spans the membrane 11 times. Two structurally similar halves span the membrane with opposite polarity. Structures with and without ammonia or methyl ammonia show a vestibule that recruits NH4+/NH3, a binding site for NH4+, and a 20 angstrom-long hydrophobic channel that lowers the NH4+ pKa to below 6 and conducts NH3. Favorable interactions for NH3 are seen within the channel and use conserved histidines. Reconstitution of AmtB into vesicles shows that AmtB conducts uncharged NH3.

https://science.sciencemag.org/content/sci/305/5690/1587.full.pdf

Mechanism of Ammonia Transport by Amt/MEP/Rh: Structure of AmtB at 1.35 Å

Recent studies of nitrogen (N) cycling in arctic tundra have indicated that inorganic N supplied to plants by mineralization is not sufficient to meet the annual requirement of N by many tundra species. Whereas N mineralization is slow in tundra soils and concentrations of inorganic N are low, these soils have large stocks of both structural and soluble organic N. In light of these observations, kinetics of absorption of three amino acids (glycine, aspartic acid, and glutamic acid) were measured in dominant vascular plant species of the four major ecosystem types in arctic Alaska and compared with concentrations of free amino acids in soils. Absorption rates were measured on roots using '4C-labeled substrates. Concentrations of free amino acids in soil were measured on water-extracted samples by high pressure liquid chromatography. All species had higher capacity (Vmax) for ammonium uptake (measured using methylamine as an ammonium analogue) than for any amino acid. However, at concentrations observed in the field, uptake rates estimated for amino acids were similar to (glycine) or less than (aspartic and glutamic acids) that for ammonium. On the basis of these comparisons, uptake rates of the three amino acids together may account for between 10 and 82% of the total N uptake in the field, depending on species and community. Deciduous shrubs had higher uptake rates than the more slowly growing evergreen shrubs, suggesting that new growth created a sink that strongly influenced capacity for amino acid uptake. In general, ectomycorrhizal species had higher amino acid uptake than did non-mycorrhizal species. In species that were sampled from more than one community, amino acid uptake rates were highest in the community where a given amino acid was most abundant in the soil. The results indicate that, in arctic tundra, plants short-circuit the mineralization step of decomposition by directly absorbing amino acids. This implies that in the organic soils of these tundra systems (1) inorganic nitrogen is an inadequate measure of plant-available soil nitrogen, (2) mineralization rates underestimate nitrogen supply rates to plants, (3) the large differences among species in capacities to absorb different forms of N provide ample basis for niche differentiation of what was previously considered a single resource, and (4) by short-circuiting N mineralization, plants accelerate N turnover and effectively exert greater control over N cycling than has been previously recognized.

Amino Acid Absorption by Arctic Plants: Implications for Plant Nutrition and Nitrogen Cycling

The relationship between growth, nutrient nitrogen assimilation, and the appearance of ligninolytic activity was examined in stationary batch cultures of the wood-destroying hymenomycete Phanerochaete chrysosporium Burds. grown under conditions optimized for lignin metabolism. A reproducible sequence of events followed inoculation: 0 to 24 h, germination, linear growth, and depletion of nutrient nitrogen; 24 to 48 h, cessation of linear growth and derepression of ammonium permease activity (demonstrating nitrogen starvation); 72 to 96 h, appearance of ligninolytic activity (synthetic 14C-lignin leads to 14CO2). Experiments with cycloheximide demonstrated that appearance of ligninolytic activity occurs irrespective of the presence of lignin; lignin did not induce additional activity. Addition of NH4+ to cultures immediately prior to the time of appearance of the ligninolytic system delayed its appearance, suggesting that the NH4+ led to interference with synthesis of the enzyme system. Addition of NH4+ to ligninolytic cultures resulted in an eventual, temporary decrease in ligninolytic activity. The results suggest that all or essential protein components of the ligninolytic enzyme system are synthesized as part of a series of physiological ("secondary metabolic") events that are initiated by nutrient nitrogen starvation.

Ligninolytic enzyme system of Phanaerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation.

In Aspergillus nidulans, mutations, designated areAr, can result in the inability to utilise a wide variety of nitrogen sources including amino acids, purines, amides, nitrate, and nitrite, whilst not affecting growth on ammonium. Other allelic areA mutations, designated areAd, lead to derepression of one or more activities which are ammonium repressible in wild type (areA+) strains, whilst not affecting their inducibility. Various areA mutations exhibit a wide variety of phenotypes: areAr alleles can be temperature sensitive on some nitrogen sources while not on others, and different alleles can be temperature sensitive for utilisation of different nitrogen sources. areAd alleles can be derepressed for one ammonium-repressible activity, be normally repressible for another, and lead to abnormally low levels for a third. Once again each areAd allele has its own highly specific phenotype. The inability of areAr strains to utilise most nitrogen sources is paralleled by low activities of certain ammonium-repressible enzymes. areAr mutations appear to be epistatic to some but not all regulatory mutations leading to constitutive synthesis of inducible enzymes and also epistatic to gdhA mutations which lead both to loss of NADP-linked glutamate dehydrogenase and to derepression of ammonium-repressible activities. 
areAr mutations do not interfere with repair of a large number of auxotrophies in double mutants. Furthermore, although areAr mutations prevent utilisation of L-arginine, L-ornithine, and L-α-amino-n-butyrate as nitrogen sources, they do not prevent the metabolism of these compounds necessary for repairing auxotrophies for proline and isoleucine in the appropriate double mutants. Utilisation of acetamide and most amino acids as carbon or carbon and nitrogen sources is unaffected by areAr mutations, and areAr strains are able to utilise acetamide and L-proline (but not other amino acids) as nitrogen sources in the presence of non-catabolite-repressing carbon sources such as L-arabinose, glycerol, melibiose, and lactose. Suppressor mutations, designated creAd, probably leading to loss of carbon catabolite repression, allow utilisation of acetamide and proline as nitrogen sources in areAr double mutants in the presence of carbon catabolite-repressing carbon sources. creAd mutations allow ethanol to serve as a source of acetate for pyruvate dehydrogenaseless (pdhA) strains in the presence of carbon catabolite-repressing carbon sources, whereas pdhA single mutants respond to ethanol as sole carbon source only in the presence of non-carbon catabolite-repressing carbon sources. Specific suppressor mutations, designated amd
d and prn
d, allow utilisation of acetamide or proline, respectively, in areAr double mutants. The areA locus can be interpreted as specifying a protein which is capable of (and in most cases essential for) allowing the synthesis of a number of enzymes of nitrogen metabolism but which cannot function in the presence of ammonium (i.e., as specifying a positive regulatory element which mediates ammonium repression) although the possibility that the areA product also plays a negative regulatory role cannot at present be ruled out.

Nitrogen metabolite repression in Aspergillus nidulans.

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however, functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH(4)(+) or NH(3)) of the transported species. We have solved the structure of wild-type AmtB from Escherichia coli in two crystal forms at 1.8- and 2.1-A resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB. At the periplasmic entry, a binding site for NH(4)(+) is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent, with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH(3.) The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transporters/rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner.

https://www.pnas.org/content/pnas/101/49/17090.full.pdf

S. Lynette Hackette

Papers

Characterization of an Ammonium Transport System in Filamentous Fungi with Methylammonium-14C As the Substrate