Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A.

Post-translational modification of P II signal transduction proteins.

The PII proteins constitute one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in all known cases they achieve their regulatory effect by direct interaction with their target.

Association and dissociation of the GlnK-AmtB complex in response to cellular nitrogen status can occur in the absence of GlnK post-translational modification.

PII proteins are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and their regulatory effect is achieved by direct interaction with their target. Many, but by no means all, PII proteins are subject to post-translational modification of a residue within the T-loop of the protein.

P(II) signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate.

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. In all these organisms, P(II) proteins coordinate many facets of nitrogen metabolism by interacting with and regulating the activities of enzymes, transcription factors, and membrane transport proteins. The primary mode of signal perception by P(II) proteins derives from their ability to bind the effector molecules 2-oxoglutarate (2-OG) and ATP or ADP.

Ammonium transport proteins with changes in one of the conserved pore histidines have different performance in ammonia and methylamine conduction.

Two conserved histidine residues are located near the mid-point of the conduction channel of ammonium transport proteins. The role of these histidines in ammonia and methylamine transport was evaluated by using a combination of in vivo studies, molecular dynamics (MD) simulation, and potential of mean force (PMF) calculations. Our in vivo results showed that a single change of either of the conserved histidines to alanine leads to the failure to transport methylamine but still facilitates good growth on ammonia, whereas double histidine variants completely lose their ability to transport both methylamine and ammonia.

P(II) signal transduction proteins: nitrogen regulation and beyond.

The P(II) proteins are one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably, P(II) proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in recent years the extent of these interactions has been recognized to be much greater than heretofore described.

PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity.

The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energy- expensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment.

Genome-wide analysis of the role of GlnR in Streptomyces venezuelae provides new insights into global nitrogen regulation in actinomycetes.

Background: GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of genes in response to changes in nitrogen availability. We applied a global in vivo approach to identify the GlnR regulon of Streptomyces venezuelae, which, unlike many actinomycetes, grows in a diffuse manner that is suitable for physiological studies. Conditions were defined that facilitated analysis of GlnR-dependent induction of gene expression in response to rapid nitrogen starvation.

The role of effector molecules in signal transduction by PII proteins.

PII proteins are one of the most widely distributed signal transduction proteins in Nature, being ubiquitous in bacteria, archaea and plants. They act by protein-protein interaction to control the activities of a wide range of enzymes, transcription factors and transport proteins, the great majority of which are involved in cellular nitrogen metabolism. The regulatory activities of PII proteins are mediated through their ability to bind the key effector metabolites 2-OG (2-oxoglutarate), ATP and ADP.

Potentials of mean force and permeabilities for carbon dioxide, ammonia, and water flux across a Rhesus protein channel and lipid membranes.

As a member of the ubiquitous ammonium transporter/methylamine permease/Rhesus (Amt/MEP/Rh) family of membrane protein channels, the 50 kDa Rhesus channel (Rh50) has been implicated in ammonia (NH(3)) and, more recently, also in carbon dioxide (CO(2)) transport. Here we present molecular dynamics simulations of spontaneous full permeation events of ammonia and carbon dioxide across Rh50 from Nitrosomonas europaea. The simulations show that Rh50 is functional in its crystallographic conformation, without the requirement for a major conformational change or the action of a protein partner.

Control of AmtB-GlnK complex formation by intracellular levels of ATP, ADP, and 2-oxoglutarate.

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. They play a major role in coordinating nitrogen metabolism by interacting with, and regulating the activities of, a variety of enzymes, transcription factors, and membrane transport proteins. The regulatory properties of P(II) proteins derive from their ability to bind three effectors: ATP, ADP, and 2-oxoglutarate.

A new P(II) protein structure identifies the 2-oxoglutarate binding site.

P(II) proteins of bacteria, archaea, and plants regulate many facets of nitrogen metabolism. They do so by interacting with their target proteins, which can be enzymes, transcription factors, or membrane proteins. A key feature of the ability of P(II) proteins to sense cellular nitrogen status and to interact accordingly with their targets is their binding of the key metabolic intermediate 2-oxoglutarate (2-OG).

Organophosphate hydrolase in Brevundimonas diminuta is targeted to the periplasmic face of the inner membrane by the twin arginine translocation pathway.

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene.

Crystal structure of dinitrogenase reductase-activating glycohydrolase (DraG) reveals conservation in the ADP-ribosylhydrolase fold and specific features in the ADP-ribose-binding pocket.

Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases.

In vitro interactions between the PII proteins and the nitrogenase regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase-activating glycohydrolase (DraG) in Azospirillum brasilense.

The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the P(II) proteins GlnB and GlnZ. We have previously shown that DraG interacts with GlnZ both in vivo and in vitro and that DraT interacts with GlnB in vivo.

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB.

The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic "gate" formed by two highly conserved phenylalanine residues, F107 and F215.

Biodegradation of aromatic compounds: an overview of meta-fission product hydrolases.

Meta fission product (MFP) hydrolases catalyze hydrolysis of a low reactive carbon-carbon bond found in meta-fission products, generated during biodegradation of various aromatic compounds. These enzymes belong to the alpha/beta hydrolase super family and show structural conservation despite having poor sequence similarity. MFP-hydrolases are substrate specific and studies have indicated that this substrate specificity plays a key role in the determination of the organism's ability to degrade a particular substrate.

The 1.3-A resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins.

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels.

Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteria.

Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the P(II) family.

Evolution and functional characterization of the RH50 gene from the ammonia-oxidizing bacterium Nitrosomonas europaea.

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an RH50 gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium Nitrosomonas europaea. Here we have used a phylogenetic approach to study the evolution of the N.

Reprint of "Structural and mechanistic aspects of Amt/Rh proteins" [J. Struct. Biol. 158 (2007) 472-481].

Amt/Rh proteins, which mediate movement of ammonium across cell membranes, are spread throughout the three kingdoms of life. Most functional studies on various members of the family have been performed using cellular assays in heterologous expression systems, which are, however, not very well suited for detailed mechanistic studies. Although now generally considered to be ammonia conducting channels, based on a number of experimental studies and structural insights, the possibility remains that some plant Amts facilitate net ammonium ion transport.

The conserved carboxy-terminal region of the ammonia channel AmtB plays a critical role in channel function.

The ammonium transport (Amt) proteins are a highly conserved family of integral membrane proteins found in eubacteria, archaea, fungi and plants. Genetic, biochemical and bioinformatic analyses suggest that they have a common tertiary structure comprising eleven trans-membrane helices with an N-out, C-in topology. The cytoplasmic C-terminus is variable in length but includes a core region of some 22 residues with considerable sequence conservation.

Structural and mechanistic aspects of Amt/Rh proteins.

Amt/Rh proteins, which mediate movement of ammonium across cell membranes, are spread throughout the three kingdoms of life. Most functional studies on various members of the family have been performed using cellular assays in heterologous expression systems, which are, however, not very well suited for detailed mechanistic studies. Although now generally considered to be ammonia conducting channels, based on a number of experimental studies and structural insights, the possibility remains that some plant Amts facilitate net ammonium ion transport.

The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel.

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the PII signal transduction protein GlnK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GlnK at a resolution of 2.