Vitamin D3 formation in milk by UV treatment - Novel insights into a rediscovered process.

Vitamin D3 is essential for several functions in the human body and the demand is usually covered by natural reactions in skin with UV radiation delivered by the sun. But living beyond a latitude of 35° can lead to a lack of sufficient exposition to the deciding wavelength. Here, many countries fortify their milk prophylactically with artificial vitamin D3. However, the precursor molecule of vitamin D3 (7-deydrocholesterol) is already naturally located in the milk fat globule membrane. Thus, this study deals with the transformation of the naturally occurring 7-dehydrocholesterol into vitamin D3 through UV treatment of the milk - a mechanism that was observed a century ago only indirectly. Different parameters such as temperature (10 - 50°C), fluid flow regimen (turbulent vs. laminar thin film, i.e., 0.6 mm) and wavelength (254, 280 and 313 nm) were investigated in this study for their efficiencies. The UV dose of each experiment was measured with chemical actinometry delivering the actually applied dose reaching the milk. Thus, the connection between applied UV dose and generated vitamin D3 content in the milk measured quantitively with LC-MS/MS was evaluated here that both were not possible a hundred years ago. The experimental results revealed that temperature generally promotes the vitamin D3 formation at 254 nm. Further, a turbulent flow is not as efficiently treated as a laminar thin film flow that is as narrow as 0.6 mm. As expected from absorbance spectra of the precursor molecule 7-dehydrocholesterol, 280 nm turned out to be the most efficient wavelength, followed by intermediate success through irradiation with 254 nm and almost no effect by 313 nm. Generally, it was shown that vitamin D3 concentration of milk was easily increased by UV treatment with today's technologies and that adjustment of certain physical parameters have a significant effect on the efficiency.

The enigmatic enzyme 'amidoxime reducing component' of Lotus japonicus. Characterization, expression, activity in plant tissues, and proposed role as a nitric oxide-forming nitrite reductase.

Human mitochondria contain a molybdoprotein capable of reducing amidoximes using cytochrome b5/cytochrome b5 reductase (Cb/CbR). This 'amidoxime reducing component' (ARC) also reduces nitrite to nitric oxide (NO). In the plant kingdom, distinct functions have been suggested for ARCs. Thus, the single ARC of Chlamydomonas reinhardtii (crARC) reduces nitrite to NO by taking electrons from nitrate reductase (NR). Therefore, it was proposed that a dual NR/crARC system can generate NO under physiological conditions and the crARC was renamed to 'NO-forming nitrite reductase' (NOFNiR). In contrast to this, the two ARC enzymes from Arabidopsis thaliana were not found to produce NO in vitro at physiological nitrite concentrations, suggesting a different, as yet unknown, function in vascular plants. Here, we have investigated the two ARCs of Lotus japonicus (LjARCs) to shed light on this controversy and to examine, for the first time, the distribution of ARCs in plant tissues. The LjARCs are localized in the cytosol and their activities and catalytic efficiencies, which are much higher than those of A. thaliana, are consistent with a role as NOFNiR. LjARCs are prone to S-nitrosylation in vitro by S-nitrosoglutathione and this post-translational modification drastically inhibits their activities. The enzymes are mainly expressed in flowers, seeds and pods, but are absent in nodules. LjARCs are active with NR and Cb/CbR as electron-transferring systems. However, the LjNR mRNA levels in seeds and pods are negligible, whereas our proteomic analyses show that pods contain the two ARCs, Cb and CbR. We conclude that LjARCs may play a role as NOFNiR by receiving electrons from the Cb/CbR system but do not act in combination with NR.

Physicochemical and Sensory Characterization of Whey Protein-Enriched Semihard Cheese.

In view of potential future changes of German food legislation with regard to cheese product quality parameters, this study aimed to evaluate the quality of whey protein-enriched semihard cheese (WPEC). Model WPEC was produced in a pilot plant and on an industrial scale by adding defined amounts of high-heat (HH) milk to the cheese milk and comprehensively analyzed during cheese processing. The dry matter, total protein, pure protein, fat, and sodium chloride content of six-week ripened cheese samples were not significantly different (p < 0.05) when the technologically necessary heating of the curd was adapted to the amount of HH milk. However, the ripening, firmness, and melting behavior of WPEC was different compared to cheese without HH milk. During ripening, no formation of whey protein peptides was observed, but differences in the amount of some bitter peptides deriving from the casein fraction were found. Sensory data suggested a slightly more bitter taste perception by the panelists for the WPEC. Further technological adjustments are recommended to obtain marketable WPEC.

Observation of a temperature dependent anomaly in the UV translucency of milk useful for UV-C preservation techniques.

Milk fat globules and casein micelles are the dispersed particles of milk that are responsible for its typical white turbid appearance and usually make it difficult to treat with modern ultraviolet light (UV) preservation techniques. The translucency of milk depends largely on the refractive indices of the dispersed particles, which are directly affected by temperature changes, as incorporated triglycerides can crystallize, melt or transition into other polymorphs. These structural changes have a significant effect on the scattering properties and thus on the UV light propagation in milk, especially by milk fat globules. In this study, a temporary minimum in the optical density of milk was observed within UV wavelength at 14 °C when heating the milk from 6 to 40 °C. This anomaly is consistent with structural changes detected by a distinct endothermic peak at 14 °C using differential scanning calorimetry. Apparently, the optical density anomaly between 10 and 20 °C disappears when the polymorphic transition already has proceeded through previous isothermal equilibration. Thus, melting of equilibrated triglycerides may not affect the RI of milk fat globules at ca. 14 °C as much as melt-mediated polymorphic transitioning. An increased efficiency of UV-C preservation (254 nm) at the translucency optimum was demonstrated by temperature-dependent microbial inactivation experiments.

Identification of Marker Peptides for the Whey Protein Quantification in Edam-Type Cheese.

Several technologies are available for incorporating whey proteins into a cheese matrix. However, there is no valid analytical method available to determine the whey protein content in matured cheese, to date. Consequently, the aim of the present study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of individual whey proteins based on specific marker peptides ('bottom-up' proteomic approach). Therefore, the whey protein-enriched model of the Edam-type cheese was produced in a pilot plant and on an industrial scale. Tryptic hydrolysis experiments were performed to evaluate the suitability of identified potential marker peptides (PMPs) for α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG). Based on the findings, α-LA and ß-LG appeared to be resistant to proteolytic degradation during six weeks of ripening and no influence on the PMP was observed. Good levels of linearity (R2 > 0.9714), repeatability (CVs < 5%), and recovery rate (80% to 120%) were determined for most PMPs. However, absolute quantification with external peptide and protein standards revealed differences in model cheese depending on the PMP, e.g., 0.50% ± 0.02% to 5.31% ± 0.25% for ß-LG. As protein spiking prior to hydrolysis revealed differing digestion behavior of whey proteins, further studies are required to enable valid quantification in various cheese types.

Uridine as a non-toxic actinometer for UV-C treatment: influence of temperature and concentration.

UV-C treatment is an effective method to inactivate microorganisms and therefore gets increasingly more attention in food industry, especially for liquid products. To test and monitor different UV-C reactor designs, a photochemical actinometer is required that gives reliable UV-C dose values and is non-toxic allowing frequent control of the production chain. Here, a variable concentrated aqueous uridine solution is tested as a photochemical actinometer. Uridine reacts at 262 nm by photohydration to a single photoproduct not absorbing any light. A concentration dependent quantum yield (Ф) was quantified in the range of 0.2-3.0 mM uridine. Results show that uridine is as accurate as the commonly accepted iodide/iodate actinometry, but not as precise. Especially at higher concentrations a higher number of measurements becomes necessary. Further, a temperature correction is presented for 10 °C > ϑ > 30 °C. Taking these results into account, uridine can certainly be considered as a non-toxic dosimeter for UV-C systems.

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 signalling pathways, Chlamydomonas ARC was shown to be a nitric oxide (NO)-forming nitrite reductase. Similar to mammals, higher plants possess two ARC proteins. To test whether plant ARCs have a similar function in NO production to the function they have in C. reinhardtii, we analysed 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 at all. 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 from Arabidopsis 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.

High-Performance Thin-Layer Chromatography-Immunostaining as a Technique for the Characterization of Whey Protein Enrichment in Edam Cheese.

Whey protein-enriched cheese can be produced by means of a high-temperature treatment of a part of the cheese milk. In this way, the nutritional quality of the resulting cheeses can be increased while resources are conserved. High-performance thin-layer chromatography-immunostaining (HPTLC-IS) using specific ß-lactoglobulin (ß-LG) antibodies was applied to study the implementation and stability of ß-LG in two different sample sets of whey protein-enriched Edam model cheeses, including industrial-scale ones. Two methods were compared for the extraction of the proteins/peptides from the cheese samples. By applying tryptic hydrolysis directly from a suspended cheese sample instead of a supernatant of a centrifuged suspension, a better yield was obtained for the extraction of ß-LG. When applying this method, it was found that selected epitopes in the tryptic ß-LG peptides remain stable over the ripening period of the cheese. For four of the tryptic ß-LG peptides detected by immunostaining, the amino acid sequence was identified using MALDI-TOF-MS/MS. One of the peptides identified was the semi-tryptic peptide VYVEELKPTP. A linear relationship was found between the content of this peptide in cheese and the proportion of high-heated milk in the cheese milk. ß-LG enrichment factors of 1.72 (n = 3, sample set I) and 1.33 ± 0.19 (n = 1, sample set II) were determined for the cheese samples containing 30% high-heated milk compared to the non-enriched samples. The relative ß-LG contents in the cheese samples with 30% high-heated milk were calculated to be 4.35% ± 0.39% (sample set I) and 9.11% ± 0.29% (sample set II) using a one-point calibration. It can be concluded that the HPTLC-IS method used is a suitable tool for the analysis of whey protein accumulation in cheese, being therefore potentially directly applicable on an industrial scale. For more accurate quantification of the whey protein content in cheese, an enhanced calibration curve needs to be applied.

The function of glutaredoxin GRXS15 is required for lipoyl-dependent dehydrogenases in mitochondria.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe-S supply in grxs15 mutants.

Isolation and Characterization of Potential Starter Cultures from the Nigerian Fermented Milk Product nono.

Nono, an important traditional fermented dairy food produced from cow's milk in Nigeria, was studied for microbial diversity and for starter culture development for industrial production. On the basis of a polyphasic approach, including phenotypic and genotypic methods such as 16S rRNA gene sequencing, repetitive element PCR (rep-PCR) fingerprinting metagenomics, and whole genome sequencing, we identified Lactobacillus (Lb.) helveticus, Limosilactobacillus (L.) fermentum, Lb. delbrueckii, and Streptococcus (S.) thermophilus as predominant bacterial species involved with milk fermentation during traditional nono production in Nigeria, while the predominant yeast species in nono was identified as Saccharomyces cerevisiae. Using metagenomics, Shigella and potential pathogens such as enterobacteria were detected at low levels of abundance. Strains of the predominant lactic acid bacteria (LAB) were selected for starter cultures combination on the basis of their capacities for rapid growth in milk and reduction of pH below 4.5 and their gelling characteristic, which was demonstrated noticeably only by the S. thermophilus strains. Whole genome sequence analysis of selected bacterial strains showed the largest assembled genome size to be 2,169,635 bp in Lb. helveticus 314, while the smallest genome size was 1,785,639 bp in Lb. delbrueckii 328M. Genes encoding bacteriocins were not detected in all the strains, but all the LAB possessed genes potentially involved in diacetyl production and citrate metabolism. These bacteria isolated from nono can thus be used to improve the microbial safety quality of nono in Nigeria, in addition to improving technological parameters such as gelling viscosity, palatability, and product consistency.

Protein oxidation during temperature-induced amyloid aggregation of beta-lactoglobulin.

Although the connection between protein oxidation, amyloid aggregation and diseases such as Alzheimer's is well known there is no information on such effects during preparation of beta-lactoglobulin fibrils. Different morphologies of amyloid aggregates of beta-lactoglobulin were prepared by incubation at pH 2 or pH 3.5 for up to 72 h. After 5 h, amyloid aggregates at pH 2 formed typical fibrils, which consisted of peptides. At pH 3.5, the amyloid aggregates were worm-like and consisted of intact protein. After 72 h, the building blocks at both pH values changed towards smaller peptides. The apparent tyrosine oxidation reached a maximum after 5 h at both pH values, whereas N-formylkynurenine and carbonyls increased continuously during 72 h. In case amyloid structures are used as edible material, the health related effects caused by protein oxidation needs to be considered.

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.

Isolation and Characterization of Lactic Acid Bacteria from Fermented Goat Milk in Tajikistan.

The lactobacilli associated with a fermented goat milk product from Tajikistan were isolated to characterize their technological properties and antibiotic resistances in order to assess their suitability for development as starter cultures. In this study, twenty three strains were identified by 16S rRNA sequencing as typical dairy-associated lactic acid bacterial strains, i.e. L. plantarum, L. pentosus, L. delbrueckii, L. helveticus and L. paracasei. These strains were generally susceptible to most antibiotics tested in this study and this allowed a selection of strains as safe starters. The draft genomes of four representative strains were sequenced and the number of contigs of the four assembled genomes ranged from 51 to 245 and the genome sizes ranged from 1.75 to 3.24 Mbp. These representative strains showed differences in their growth behavior and pH-reducing abilities in in vitro studies. The co-inoculation of these Lactobacillus spp. strains together with a yeast Kluyveromyces marxianus MBT-5698, or together with the yeast and an additional Streptococcus thermophilus MBT-2, led to a pH reduction to 3.4 after 48 h. Only in the case of fermentation inoculated with the co-culture, the viscosity of the milk increased noticeably. In contrast, fermentations with single strains did not lead to gelation of the milk or to a decrease in the pH after 24h. The results of this study provide a comprehensive understanding of the predominant lactobacilli related to Tajikistani fermented milk products.

Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni.

Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s-1 and a KM (nitrate) of 3.40 ± 0.44 µM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals.

The evolutionarily highly conserved SNF1-related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth- and stress response-related metabolic pathways. While the regulation of the mammalian [AMP-activated protein kinase (AMPK)] and yeast (SNF1) orthologues of SnRK1 is well-characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T-loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions.

Cellular Response to Titanium Dioxide Nanoparticles in Intestinal Epithelial Caco-2 Cells is Dependent on Endocytosis-Associated Structures and Mediated by EGFR.

Titanium dioxide (TiO2) is one of the most applied nanomaterials and widely used in food and non-food industries as an additive or coating material (E171). It has been shown that E171 contains up to 37% particles which are smaller than 100 nm and that TiO2 nanoparticles (NPs) induce cytotoxicity and inflammation. Using a nuclear factor Kappa-light-chain enhancer of activated B cells (NF-κB) reporter cell line (Caco-2nfkb-RE), Real time polymerase chain reaction (PCR), and inhibition of dynamin and clathrin, it was shown that cellular responses induced by 5 nm and 10 nm TiO2 NPs (nominal size) depends on endocytic processes. As endocytosis is often dependent on the epithelial growth factor receptor (EGFR), further investigations focused on the involvement of EGFR in the uptake of TiO2 NPs: (1) inhibition of EGFR reduced inflammatory markers of the cell (i.e., nuclear factor (NF)-κB activity, mRNA of IL8, CCL20, and CXCL10); and (2) exposure of Caco-2 cells to TiO2 NPs activated the intracellular EGFR cascade beginning with EGFR-mediated extracellular signal-regulated kinases (ERK)1/2, and including transcription factor ELK1. This was followed by the expression of ERK1/2 target genes CCL2 and CXCL3. We concluded that TiO2 NPs enter the cell via EGFR-associated endocytosis, followed by activation of the EGFR/ERK/ELK signaling pathway, which finally induces NF-κB. No changes in inflammatory response are observed in Caco-2 cells exposed to 32 nm and 490 nm TiO2 particles.

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 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω 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 14-3-3ω to confer full inhibition of NR activity under physiological conditions.

Sulfite Oxidase Catalyzes Single-Electron Transfer at Molybdenum Domain to Reduce Nitrite to Nitric Oxide.

AIMS: Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, and mARC exhibit nitrite reductase activity at low oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent changes in blood flow, leading to continued exploration for other candidate nitrite reductases. Another physiologically important molybdenum enzyme­sulfite oxidase (SO)­has not been extensively studied. RESULTS: Using gas-phase nitric oxide (NO) detection and physiological concentrations of nitrite, SO functions as nitrite reductase in the presence of a one-electron donor, exhibiting redox coupling of substrate oxidation and nitrite reduction to form NO. With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Studies with recombinant heme and molybdenum domains of SO indicate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain, mediated by steric and redox effects of this domain. Using knockdown of all molybdopterin enzymes and SO in fibroblasts isolated from patients with genetic deficiencies of molybdenum cofactor and SO, respectively, SO was found to significantly contribute to hypoxic nitrite signaling as demonstrated by activation of the canonical NO-sGC-cGMP pathway. INNOVATION: Nitrite binds to and is reduced at the molybdenum site of mammalian SO, which may be allosterically regulated by heme and molybdenum domain interactions, and contributes to the mammalian nitrate-nitrite-NO signaling pathway in human fibroblasts. CONCLUSION: SO is a putative mammalian nitrite reductase, catalyzing nitrite reduction at the Mo(IV) center.

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.

Structural basis of thermal stability of the tungsten cofactor synthesis protein MoaB from Pyrococcus furiosus.

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 Pyrococcus furiosus, 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.