Characterization of the amidoxime reducing components ARC1 and ARC2 from Arabidopsis thaliana

Five molybdenum-dependent enzymes are known in eukaryotes. While four of them are under investigation since decades, the most recently discovered, (mitochondrial) amidoxime reducing component ((m)ARC), has only been characterized in mammals and the green algae Chlamydomonas reinhardtii. While mammalian mARCs have been shown to be involved in various signaling pathways, Chlamydomonas ARC was shown to be a nitric oxide (NO)-forming nitrite reductase. Similar to mammals, higher plants possess two ARC proteins. In order to test whether plant ARCs have a similar function in NO production to the function they have in C. reinhardtii, we analyzed the enzymes from the model plant Arabidopsis thaliana. Both ARC1 and ARC2 from Arabidopsis could reduce N-hydroxylated compounds, while nitrite reduction to form NO could only be demonstrated for ARC2. Searching for physiological electron donors we found that both ARC enzymes accept electrons from NADH via cytochrome b5 reductase and cytochrome b5, but only ARC2 is able to accept electrons from nitrate reductase. Furthermore, arc-deficient mutant plants were similar to wildtype plants regarding growth and also nitrite-dependent NO-formation. Altogether, our results did not confirm the hypothesis that either ARC1 or ARC2 are involved in physiologically relevant nitrite-dependent NO-formation. In contrast, our data suggest that ARC1 and ARC2 have distinct, yet unknown physiological roles in higher plants

Isoform-Specific NO Synthesis by Arabidopsis thaliana Nitrate Reductase

Nitrate reductase (NR) is important for higher land plants, as it catalyzes the rate-limiting step in the nitrate assimilation pathway, the two-electron reduction of nitrate to nitrite. Furthermore, it is considered to be a major enzymatic source of the important signaling molecule nitric oxide (NO), that is produced in a one-electron reduction of nitrite. Like many other plants, the model plant Arabidopsis thaliana expresses two isoforms of NR (NIA1 and NIA2). Up to now, only NIA2 has been the focus of detailed biochemical studies, while NIA1 awaits biochemical characterization. In this study, we have expressed and purified functional fragments of NIA1 and subjected them to various biochemical assays for comparison with the corresponding NIA2-fragments. We analyzed the kinetic parameters in multiple steady-state assays using nitrate or nitrite as substrate and measured either substrate consumption (nitrate or nitrite) or product formation (NO). Our results show that NIA1 is the more efficient nitrite reductase while NIA2 exhibits higher nitrate reductase activity, which supports the hypothesis that the isoforms have special functions in the plant. Furthermore, we successfully restored the physiological electron transfer pathway of NR using reduced nicotinamide adenine dinucleotide (NADH) and nitrate or nitrite as substrates by mixing the N-and C-terminal fragments of NR, thus, opening up new possibilities to study NR activity, regulation and structure

A sensitive and stable amperometric nitrate biosensor employing Arabidopsis thaliana nitrate reductase

Nitrate reductase (NR) from the plant Arabidopsis thaliana has been employed in the development of an amperometric nitrate biosensor that functions at physiological pH. The anion anthraquinone-2-sulfonate (AQ) is used as an effective artificial electron transfer partner for NR at a glassy carbon (GC) electrode. Nitrate is enzymatically reduced to nitrite and the oxidized form of NR is electrochemically reduced by the hydroquinone form of the mediator (AQH2). The GC/NR electrode shows a pronounced cathodic wave for nitrate reduction and the catalytic current increases linearly in the nitrate concentration range of 10-400 μM with a correlation coefficient of 0.989. Using an amperometric method, a low detection limit of 0.76 nM (S/N = 3) was achieved. The practical application of the present electrochemical biosensor was demonstrated by the determination of nitrate concentration in natural water samples and the results agreed well with a standard spectroscopic method. Graphical Abstract: [Figure not available: see fulltext.

Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity

14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3 omega to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3 omega variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3 omega to confer full inhibition of NR activity under physiological conditions

Mediated electrochemistry of nitrate reductase from Arabidopsis thaliana

Herein we report the mediated electrocatalytic voltammetry of the plant molybdoenzyme nitrate reductase (NR) from Arabidopsis thaliana using the established truncated molybdenum-heme fragment at a glassy carbon (GC) electrode. Methyl viologen (MV), benzyl viologen (BV), and anthraquinone-2- sulfonic acid (AQ) are employed as effective artificial electron transfer partners for NR, differing in redox potential over a range of about 220 mV and delivering different reductive driving forces to the enzyme. Nitrate is reduced at the Mo active site of NR, yielding the oxidized form of the enzyme, which is reactivated by the electro-reduced form of the mediator. Digital simulation was performed using a single set of enzyme dependent parameters for all catalytic voltammetry obtained under different sweep rates and various substrate or mediator concentrations. The kinetic constants from digital simulation provide new insight into the kinetics of the NR catalytic mechanism

Structural Basis of Thermal Stability of the Tungsten Cofactor Synthesis Protein MoaB from <i>Pyrococcus furiosus</i>

<div><p>Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism <i>Pyrococcus furiosus</i>, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15°C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50°C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.</p></div

The Crystal Structure of Plant Sulfite Oxidase Provides Insights into Sulfite Oxidation in Plants and Animals

AbstractThe molybdenum cofactor (Moco) containing sulfite oxidase (SO) from Arabidopsis thaliana has recently been identified and biochemically characterized. The enzyme is found in peroxisomes and believed to detoxify excess sulfite that is produced during sulfur assimilation, or due to air pollution. Plant SO (PSO) is homodimeric and homologous to animal SO, but contains only a single Moco domain without an additional redox center. Here, we present the first crystal structure of a plant Moco enzyme, the apo-state of Arabidopsis SO at 2.6 Å resolution. The overall fold and coordination of the Moco are similar to chicken SO (CSO). Comparisons of conserved surface residues and the charge distribution in PSO and CSO reveal major differences near the entrance to both active sites reflecting different electron acceptors. Arg374 has been identified as an important substrate binding residue due to its conformational change when compared to the sulfate bound structure of CSO

Biochemical characterization of the PfuMoaB-H3 variant in comparison to PfuMoaB-WT.

<p>(A) 15% Coomassie-Blue-stained SDS polyacrylamide gel showing 200 pmol of Ni-NTA-purified PfuMoaB-WT and PfuMoaB-H3. (B) Far-UV CD spectra of Ni-NTA purified PfuMoaB-WT (solid line) and PfuMoaB-H3 (dotted line). (C) Size exclusion chromatography of Ni-NTA purified PfuMoaB-WT and PfuMoaB-H3. 5 nmol of WT and 10 nm of PfuMoaB-H3 were applied on a Superdex 200 10/300 column. Peaks referring to the different oligomerization states of both proteins are labelled. Molecular masses were determined using protein standards. Elution of PfuMoaB-WT is shown as solid line, the PfuMoaB-H3 variant as dotted line. (D–E) SDS-PAGE of cross-linked PfuMoaB-WT and PfuMoaB-H3 with BS³ (D) and EDC (E). Samples without addition of cross-linkers were used as control (“–”). Observed oligomeric forms of both proteins are labelled. The cross-linked protein bands with a size corresponding to the trimers (designated with *) were further subjected to mass spectrometry analysis. (F) Differential scanning calorimetry of MPT-adenylyl-transferases. Melting curves of PfuMoaB-WT, PfuMoaB-H3, EcoMogA, EcoMoaB and AthCnx1G recorded by DSC. The maximum of each peak represents the respective <i>T</i>_m value. Average <i>T</i>_m values for each protein are summarized in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone-0086030-t002" target="_blank">Table 2</a>. Measurements were performed in duplicate for each experiment.</p

Active site of PfuMoaB-WT.

<p>Two PfuMoaB subunits at the hexamerization interface are shown as ribbon in green and grey, respectively. The conserved Asp56 residue coordinating Mg²⁺ (pink) is shown in sticks. MPT-AMP in the active site is derived from a superimposition with the structure of the PfuMoaB homologue <i>A. thaliana</i> Cnx1G (1UUY). The Mg²⁺-ion derived from a superimposition with the homologues sub-domain 3 of <i>E. coli</i> MoeA (1FC5) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone.0086030-Kuper1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone.0086030-Schrag1" target="_blank">[63]</a>.</p