scholarly journals The Oxoglutarate Binding Site and Regulatory Mechanism Are Conserved in Ammonium Transporter Inhibitors GlnKs from Methanococcales

2021 ◽  
Vol 22 (16) ◽  
pp. 8631
Author(s):  
Marie-Caroline Müller ◽  
Tristan Wagner
Keyword(s):  
Energy Charge ◽  
Methanogenic Archaea ◽  
Adaptive Strategy ◽  
Terminal Group ◽  
Ammonium Transporter ◽  
Methanocaldococcus Jannaschii ◽  
Protein Inhibition ◽  
Binding Interface ◽  
Ammonium Transporters ◽  
Carboxy Terminal

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK–Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

scholarly journals S-Inosyl-l-Homocysteine Hydrolase, a Novel Enzyme Involved inS-Adenosyl-l-Methionine Recycling

2015 ◽  
Vol 197 (14) ◽  
pp. 2284-2291 ◽  
Author(s):  
Danielle Miller ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Metabolic Pathways ◽  
Methanogenic Archaea ◽  
Metabolite Identification ◽  
Strictly Anaerobic ◽  
Methanocaldococcus Jannaschii ◽  
Content Type ◽  
Sequence Identity ◽  
Feedback Inhibitor ◽  
Recycling Pathway ◽  
Insight Into

ABSTRACTS-Adenosyl-l-homocysteine, the product ofS-adenosyl-l-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific forS-adenosyl-l-homocysteine producesl-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotatedS-adenosyl-l-homocysteine hydrolase inMethanocaldococcus jannaschiiis specific for the hydrolysis and synthesis ofS-inosyl-l-homocysteine, notS-adenosyl-l-homocysteine. This is the first report of an enzyme specific forS-inosyl-l-homocysteine. As withS-adenosyl-l-homocysteine hydrolase, which shares greater than 45% sequence identity with theM. jannaschiihomologue, theM. jannaschiienzyme was found to copurify with bound NAD+and hasKmvalues of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11 mM for inosine,l-homocysteine, andS-inosyl-l-homocysteine, respectively. No enzymatic activity was detected withS-adenosyl-l-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate theM. jannaschiienzyme anS-inosyl-l-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM fromS-adenosyl-l-homocysteine that uses the deamination ofS-adenosyl-l-homocysteine to formS-inosyl-l-homocysteine.IMPORTANCEIn strictly anaerobic methanogenic archaea, such asMethanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway forS-adenosyl-l-homocysteine, produced fromS-adenosyl-l-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific forS-inosyl-l-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the biochemical reactions that were occurring when life originated.

scholarly journals Differential Activation of Ammonium Transporters During the Accumulation of Ammonia by Colletotrichum gloeosporioides and Its Effect on Appressoria Formation and Pathogenicity

2013 ◽  
Vol 26 (3) ◽  
pp. 345-355 ◽  
Author(s):  
Chen Shnaiderman ◽  
Itay Miyara ◽  
Ilana Kobiler ◽  
Amir Sherman ◽  
Dov Prusky
Keyword(s):  
Gene Disruption ◽  
Colletotrichum Gloeosporioides ◽  
Ammonium Transporter ◽  
Ammonium Transport ◽  
Wild Type ◽  
Ammonia Accumulation ◽  
Ammonium Transporters ◽  
Avocado Fruit ◽  
Appressoria Formation ◽  
Encoding Genes

Ammonium secreted by the post-harvest pathogen Colletotrichum gloeosporioides during host colonization accumulates in the host environment due to enhanced fungal nitrogen metabolism. Two types of ammonium transporter-encoding genes, AMET and MEP, are expressed during pathogenicity. Gene disruption of AMET, a gene modulating ammonia secretion, showed twofold reduced ammonia secretion and 45% less colonization on avocado fruit, suggesting a contribution to pathogenicity. MEPB, a gene modulating ammonium transport, is expressed by C. gloeosporioides during pathogenicity and starvation conditions in culture. Gene disruption of MEPB, the most highly expressed gene of the MEP family, resulted in twofold overexpression of MEPA and MEPC but reduced colonization, suggesting MEPB expression's contribution to pathogenicity. Analysis of internal and external ammonia accumulation by ΔmepB strains in mycelia and germinated spores showed rapid uptake and accumulation, and reduced secretion of ammonia in the mutant versus wild-type (WT) strains. Ammonia uptake by the WT germinating spores but not by the ΔmepB strain with compromised ammonium transport activated cAMP and transcription of PKA subunits PKAR and PKA2. ΔmepB mutants showed 75% less appressorium formation and colonization than the WT, which was partially restored by 10 mM exogenous ammonia. Thus, whereas both AMET and MEPB genes modulate ammonia secretion, only MEPB contributes to ammonia accumulation by mycelia and germinating spores that activate the cAMP pathways, inducing the morphogenetic processes contributing to C. gloeosporioides pathogenicity.

Studies on the High-Sulfur Proteins of Reduced Merino Wool

1969 ◽  
Vol 39 (10) ◽  
pp. 912-917 ◽  
Author(s):  
L. S. Swart ◽  
T. Haylett ◽  
F. J. Joubert
Keyword(s):  
Molecular Weight ◽  
Free Amino ◽  
Molecular Size ◽  
Terminal Group ◽  
Terminal Residue ◽  
Merino Wool ◽  
Amino Terminal ◽  
Carboxy Terminal

A reinvestigation of a fraction of the high-sulfur proteins SCMKB was made. The proteins were successively fractionated on an electrophoretic, molecular-size, and chromatographic basis. This work resulted in the isolation of a homogeneous protein SCMKB-IIIB2 which has a molecular weight of 11,260, no free amino-terminal group, and contains S-carboxymethyl cysteine as carboxy-terminal residue.

scholarly journals High-affinity ammonium transport by Arabidopsis thaliana AMT1;4

2021 ◽  
Vol 43 (4) ◽  
Author(s):  
Nino Bindel ◽  
Benjamin Neuhäuser
Keyword(s):  
Arabidopsis Thaliana ◽  
Xenopus Laevis ◽  
Pollen Tube ◽  
Pollen Grains ◽  
Transport Proteins ◽  
Ammonium Transporter ◽  
Ammonium Transport ◽  
High Affinity ◽  
Ammonium Transporters ◽  
Unique Expression Pattern

AbstractIn plants high affinity transport proteins mediate the essential transport of ammonium across membranes. In Arabidopsis thaliana six of these AMmonium Transporters (AMTs) are encoded by the genome. All of them show a unique expression pattern. While most AMTs are highly expressed in the root, AtAMT1;4 expression is limited to the pollen grains and the pollen tube. Here, we addressed the transport characteristics of AtAMT1;4 in the heterologous Xenopus laevis oocytes system. The transport saturated and showed high affinity for ammonium with a Km value lower than 10 µM. Based on our electrophysiological analysis, we classified AtAMT1;4 as a high affinity ammonium transporter.

scholarly journals Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 fromMethanocaldococcus jannaschii

2014 ◽  
Vol 70 (11) ◽  
pp. 1472-1479 ◽  
Author(s):  
Thomas A. Bobik ◽  
Erick J. Morales ◽  
Annie Shin ◽  
Duilio Cascio ◽  
Michael R. Sawaya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Greenhouse Gas ◽  
Active Site ◽  
Methanogenic Archaea ◽  
Anomalous Scattering ◽  
Biosynthetic Enzyme ◽  
Mechanistic Studies ◽  
Methanocaldococcus Jannaschii ◽  
C1 Metabolism ◽  
Tim Barrel

Prior studies have indicated that MJ1099 fromMethanocaldococcus jannaschiihas roles in the biosynthesis of tetrahydromethanopterin and methanofuran, two key cofactors of one-carbon (C1) metabolism in diverse organisms including the methanogenic archaea. Here, the structure of MJ1099 has been solved to 1.7 Å resolution using anomalous scattering methods. The results indicate that MJ1099 is a member of the TIM-barrel superfamily and that it is a homohexamer. Bioinformatic analyses identified a potential active site that is highly conserved among MJ1099 homologs and the key amino acids involved were identified. The results presented here should guide further studies of MJ1099 including mechanistic studies and possibly the development of inhibitors that target the methanogenic archaea in the digestive tracts of humans and that are a source of the greenhouse gas methane.

scholarly journals Different Transport Mechanisms in Plant and Human AMT/Rh-type Ammonium Transporters

2006 ◽  
Vol 127 (2) ◽  
pp. 133-144 ◽  
Author(s):  
Maria Mayer ◽  
Gabriel Schaaf ◽  
Isabelle Mouro ◽  
Claude Lopez ◽  
Yves Colin ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Alkali Cation ◽  
Ammonium Transporter ◽  
Ammonium Transport ◽  
Transport Mechanisms ◽  
Gas Channel ◽  
Yeast Strains ◽  
Cation Conductance ◽  
Ammonium Transporters ◽  
Rh Proteins

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH3 gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with Km = 3.8 mM at pHo 7.5. Uptake was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH4+ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pHo-independent MeA+ uptake and specific NH4+ and MeA+ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH4+, or cotransports NH3/H+, RhCG mediates electroneutral NH3 transport.

In-season expression of nitrate and ammonium transporter genes in roots of winter wheat (Triticum aestivum L.) genotypes with different nitrogen-uptake efficiencies

2015 ◽  
Vol 66 (7) ◽  
pp. 671 ◽  
Author(s):  
Jinshan Liu ◽  
Jie Fu ◽  
Hui Tian ◽  
Yajun Gao
Keyword(s):  
Temporal Dynamics ◽  
N Uptake ◽  
Expression Patterns ◽  
Genotypic Variation ◽  
Grain Filling ◽  
Triticum Aestivum L ◽  
Ammonium Transporter ◽  
Temporal Expression ◽  
Reverse Transcription Pcr ◽  
Ammonium Transporters

Although nitrate and ammonium transporter genes of wheat have been cloned, little is known about their expression characteristics. A greenhouse experiment was established to study temporal expression patterns over a growing season for four nitrate-transporter genes (TaNRT2.1, TaNRT2.2, TaNRT2.3 and TaNRT1.2) and two ammonium-transporter genes (TaAMT1.1 and TaAMT1.2) in wheat genotypes with different nitrogen (N)-uptake efficiencies. Genotypes that were N-efficient (XY107) and N-inefficient (XY6) were planted in soils that were N-limited (no N added) and N-adequate (added 0.3 g N kg–1 soil). Roots were sampled at tillering, jointing, heading and grain-filling stages, and the expression of the six genes was quantified using real-time, reverse transcription PCR (polymerase chain reaction). Results indicated that maintaining active N uptake during reproduction was the main strategy used by genotype XY107 to sustain its high N-uptake efficiencies in both N treatments. The expression of all NRT and AMT genes showed significant temporal dynamics, and generally matched the pattern of in-season N uptake of wheat plants. Several NRT or AMT genes (especially TaNRT2.1) showed greater expression at reproduction in the N-efficient genotype, XY107, than in the N-inefficient genotype, XY6, suggesting that nitrate and ammonium transporters play important roles in determining the genotypic variation of N uptake in wheat.

scholarly journals Interactions of the α3β2 Nicotinic Acetylcholine Receptor Interfaces with α-Conotoxin LsIA and its Carboxylated C-terminus Analogue: Molecular Dynamics Simulations

Marine Drugs ◽  
2020 ◽  
Vol 18 (7) ◽  
pp. 349
Author(s):  
Jierong Wen ◽  
David J. Adams ◽  
Andrew Hung
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Neurological Diseases ◽  
Nicotinic Acetylcholine ◽  
Binding Interface ◽  
C Terminus ◽  
Dynamics Simulations ◽  
The Individual ◽  
Carboxy Terminal ◽  
Subtype Selective

Notably, α-conotoxins with carboxy-terminal (C-terminal) amidation are inhibitors of the pentameric nicotinic acetylcholine receptors (nAChRs), which are therapeutic targets for neurological diseases and disorders. The (α3)2(β2)3 nAChR subunit arrangement comprises a pair of α3(+)β2(−) and β2(+)α3(−) interfaces, and a β2(+)β2(−) interface. The β2(+)β2(−) interface has been suggested to have higher agonist affinity relative to the α3(+)β2(−) and β2(+)α3(−) interfaces. Nevertheless, the interactions formed by these subunit interfaces with α-conotoxins are not well understood. Therefore, in order to address this, we modelled the interactions between α-conotoxin LsIA and the α3β2 subtype. The results suggest that the C-terminal carboxylation of LsIA predominantly influenced the enhanced contacts of the conotoxin via residues P7, P14 and C17 on LsIA at the α3(+)β2(−) and β2(+)α3(−) interfaces. However, this enhancement is subtle at the β2(+)β2(−) site, which can compensate the augmented interactions by LsIA at α3(+)β2(−) and β2(+)α3(−) binding sites. Therefore, the divergent interactions at the individual binding interface may account for the minor changes in binding affinity to α3β2 subtype by C-terminal carboxylation of LsIA versus its wild type, as shown in previous experimental results. Overall, these findings may facilitate the development of new drug leads or subtype-selective probes.

scholarly journals Ribose-5-Phosphate Biosynthesis in Methanocaldococcus jannaschii Occurs in the Absence of a Pentose-Phosphate Pathway

2005 ◽  
Vol 187 (21) ◽  
pp. 7382-7389 ◽  
Author(s):  
Laura L. Grochowski ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Recent Work ◽  
Pentose Phosphate Pathway ◽  
Methanogenic Archaea ◽  
Protein Product ◽  
Pentose Phosphate ◽  
The Other ◽  
Methanocaldococcus Jannaschii ◽  
Cell Extracts ◽  
Ribulose Monophosphate Pathway ◽  
Sugar Phosphates

ABSTRACT Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-2H2]- and [U-13C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of d-arabino-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the d-ara-bino-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from d-arabino-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.

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