How sensor Amt-like proteins integrate ammonium signals.

Unlike aquaporins or potassium channels, ammonium transporters (Amts) uniquely discriminate ammonium from potassium and water. This feature has certainly contributed to their repurposing as ammonium receptors during evolution. Here, we describe the ammonium receptor Sd-Amt1, where an Amt module connects to a cytoplasmic diguanylate cyclase transducer module via an HAMP domain.

The Cofactors of Nitrogenases.

In biological nitrogen fixation, the enzyme nitrogenase mediates the reductive cleavage of the stable triple bond of gaseous N2at ambient conditions, driven by the hydrolysis of ATP, to yield bioavailable ammonium (NH4+). At the core of nitrogenase is a complex, ironsulfur based cofactor that in most variants of the enzyme contains an additional, apical heterometal (Mo or V), an organic homocitrate ligand coordinated to this heterometal, and a unique, interstitial carbide. Recent years have witnessed fundamental advances in our understanding of the atomic and electronic structure of the nitrogenase cofactor.

The Critical E State of Nitrogenase Catalysis.

The reaction catalyzed by the nitrogenase enzyme involves breaking the stable triple bond of the dinitrogen molecule and is consequently considered among the most challenging reactions in biology. While many aspects regarding its atomic mechanism remain to be elucidated, a kinetic scheme established by David Lowe and Roger Thorneley has remained a gold standard for functional studies of the enzyme for more than 30 years. Recent three-dimensional structures of ligand-bound states of molybdenum- and vanadium-dependent nitrogenases have revealed the actual site of substrate binding on the large active site cofactors of this class of enzymes.

A bound reaction intermediate sheds light on the mechanism of nitrogenase.

Reduction of N by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear.

Signaling ammonium across membranes through an ammonium sensor histidine kinase.

Sensing and uptake of external ammonium is essential for anaerobic ammonium-oxidizing (anammox) bacteria, and is typically the domain of the ubiquitous Amt/Rh ammonium transporters. Here, we report on the structure and function of an ammonium sensor/transducer from the anammox bacterium "Candidatus Kuenenia stuttgartiensis" that combines a membrane-integral ammonium transporter domain with a fused histidine kinase. It contains a high-affinity ammonium binding site not present in assimilatory Amt proteins.

Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date.

The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction.

The copper-containing enzyme nitrous oxide reductase (NOR) catalyzes the transformation of nitrous oxide (NO) to dinitrogen (N) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites Cu and Cu, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes NO respiration.

Nitrogenase FeMoco investigated by spatially resolved anomalous dispersion refinement.

The [Mo:7Fe:9S:C] iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation. Only recently its atomic structure was clarified, while its reactivity and electronic structure remain under debate. Here we show that for its resting S=3/2 state the common iron oxidation state assignments must be reconsidered.

Active sites without restraints: high-resolution analysis of metal cofactors.

For most three-dimensional structures of biological macromolecules, the factual accuracy of atom positions by far exceeds the resolution of the experimental data, although the refinement problem presented by a protein structure is substantially underdetermined. This is achieved through using restraints that precisely define protein geometries and thus reduce the degrees of freedom of the refinement problem. If such information is not available or when unusual geometries or particular ligand states complicate structural analysis, possible pitfalls arise that not only concern the precise definition of spatial arrangements, but also the identification of atom types and bond distances.

Extended reaction scope of thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase: from C-C bond cleavage to C-C bond ligation.

ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) catalyzes the CC bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate. One of the two reactivities of CDH was selectively knocked down by mutation experiments. CDH-H28A is much less able to catalyze the CC bond formation, while the ability for CC bond cleavage is still intact.

Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins.

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes.

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin.

Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee).

The sixteenth iron in the nitrogenase MoFe protein.

Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca(2+) or Mg(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.

The tricky task of nitrate/nitrite antiport.

Subtle differences: Two recent crystal structures have provided the first insight into nitrate/nitrite exchangers (example shown with bound nitrite), which are crucial to bacterial metabolism. A direct comparison of the structures reveals how the proteins can distinguish between their highly similar substrates and translate this into a conformational change to translocate ions across the membrane.

Analysis of the magnetic properties of nitrogenase FeMo cofactor by single-crystal EPR spectroscopy.

The catalytic center of nitrogenase, the [Mo:7Fe:9S:C]:homocitrate FeMo cofactor, is a S=3/2 system with a rhombic magnetic g tensor. Single-crystal EPR spectroscopy in combination with X-ray diffraction were used to determine the relative orientation of the g tensor with respect to the cluster structure. The protein environment influences the electronic structure of the FeMo cofactor, dictating preferred orientations of possible functional relevance.

The formate/nitrite transporter family of anion channels.

The formate/nitrite transporter (FNT) family of integral membrane proteins comprises pentameric channels for monovalent anions that exhibit a broad specificity for small anions such as chloride, the physiological cargo molecules formate, nitrite, and hydrosulfide, and also larger organic acids. Three-dimensional structures are available for the three known subtypes, FocA, NirC, and HSC, which reveal remarkable evolutionary optimizations for the respective physiological context of the channels. FNT channels share a conserved translocation pathway in each protomer, with a central hydrophobic cavity that is separated from both sides of the membrane by a narrow constriction.

α-Hydroxy-β-keto acid rearrangement-decarboxylation: impact on thiamine diphosphate-dependent enzymatic transformations.

The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-β-keto acid rather than a donor-acceptor substrate role variation.

Structural and functional characterization of the nitrite channel NirC from Salmonella typhimurium.

Nitrite (NO(2)(-)) is a central intermediate in the nitrogen metabolism of microorganisms and plants, and is used as a cytotoxin by macrophages as part of the innate immune response. The bacterial membrane protein NirC acts as a specific channel to facilitate the transport of nitrite anions across lipid bilayers for cytoplasmic detoxification. Despite NirC's importance in nitrogen metabolism and in the pathogenicity of enteric bacteria, available biochemical data are scarce.

The formate channel FocA exports the products of mixed-acid fermentation.

Formate is a major metabolite in the anaerobic fermentation of glucose by many enterobacteria. It is translocated across cellular membranes by the pentameric ion channel/transporter FocA that, together with the nitrite channel NirC, forms the formate/nitrite transporter (FNT) family of membrane transport proteins. Here we have carried out an electrophysiological analysis of FocA from Salmonella typhimurium to characterize the channel properties and assess its specificity toward formate and other possible permeating ions.

Thermodynamics of transport through the ammonium transporter Amt-1 investigated with free energy calculations.

Amt-1 from Archaeoglobus fulgidus (AfAmt-1) belongs to the Amt/Rh family of ammonium/ammonia transporting membrane proteins. The transport mode and the precise microscopic permeation mechanism utilized by these proteins are intensely debated. Open questions concern the identity of the transported substrate (ammonia and/or ammonium) and whether the transport is passive or active.

Evidence for interstitial carbon in nitrogenase FeMo cofactor.

The identity of the interstitial light atom in the center of the FeMo cofactor of nitrogenase has been enigmatic since its discovery. Atomic-resolution x-ray diffraction data and an electron spin echo envelope modulation (ESEEM) analysis now provide direct evidence that the ligand is a carbon species.