Dimethylsulfide dehydrogenase from rhodovulum sulfidophilum: EPR spectroscopy and biochemical analysis reveal its place in the DMSO reductase family of molybdenum enzymes

Dimethylsulfide (DMS) dehydrogenase catalyses the oxidation of DMS to dimethylsulfoxide. The purified enzyme has three subunits of Mr = 94, 38 and 32 kDa and has an optical spectrum dominated by a b-type cytochrome. The metal ion and nucleotide analysis revealed 0.5 g-atom Mo, 9.8 g-atom Fe and 1.96 mol GMP per tool of enzyme. Taken together, these data indicate that DMS dehydrogenase contains a bis(MGD)Mo cofactor. A comparison of the Nterminal amino acid sequence of DMS dehydrogenase revealed that the Mo-containing ct-subunit was most closely related to the c~-subunits of nitrate reductase (NarG) and selenate reductase (SerA). Similarly, the [~-subunit of DMS dehydrogenase was most closely related to the [3-subunits of nitrate reductase (NarH) and selenate reductase (SerB). Variable temperature X-band EPR spectra (120-2K) of 'as isolated' DMS dehydrogenase showed resonances arising from multiple redox centres, Mo(V), [3Fe-4S] +, [4Fe-4S] ÷. A pH dependent EPR study of the Mo(V) centre in lH20 and 2H20 reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). The spin Hamiltonian parameters (2.0158, 1.8870, 1.8620) for the [4Fe-4S] + cluster suggests the presence of histidine (N) coordination to iron in this cluster. It is suggested that this unusual [Fe-S] cluster may be associated with a histidine-cysteine rich sequence at the N-terminus of the ct-subunit of DMS dehydrogenase.

Nitrogen ecophysiology of Heron Island, a subtropical coral cay of the Great Barrier Reef, Australia

Coral cays form part of the Australian Great Barrier Reef. Coral cays with high densities of seabirds are areas of extreme nitrogen (N) enrichment with deposition rates of up to 1000 kg N ha(-1) y(-1). The ways in which N sources are utilised by coral cay plants, N is distributed within the cay, and whether or not seabird-derived N moves from cay to surrounding marine environments were investigated. We used N metabolite analysis, N-15 labelling and N-15 natural abundance (delta(15)N) techniques. Deposited guano-derived uric acid is hydrolysed to ammonium (NH4+) and gaseous ammonia (NH3). Ammonium undergoes nitrification, and nitrate (NO3-) and NH4+ were the main forms of soluble N in the soil. Plants from seabird rookeries have a high capacity to take up and assimilate NH4+, are able to metabolise uric acid, but have low rates of NO3- uptake and assimilation. We concluded that NH4+ is the principal source of N for plants growing at seabird rookeries, and that the presence of NH4+ in soil and gaseous NH3 in the atmosphere inhibits assimilation of NO3-, although NO3- is taken up and stored. Seabird guano, Pisonia forest soil and vegetation were similarly enriched in N-15 suggesting that the isotopic enrichment of guano (delta(15)N 9.9parts per thousand) carries through the forest ecosystem. Soil and plants from woodland and beach environments had lower delta(15)N (average 6.5parts per thousand) indicating a lower contribution of bird-derived N to the N nutrition of plants at these sites. The aquifer under the cay receives seabird-derived N leached from the cay and has high concentrations of N-15-enriched NO3- (delta(15)N 7.9parts per thousand). Macroalgae from reefs with and without seabirds had similar delta(15)N values of 2.0-3.9parts per thousand suggesting that reef macroalgae do not utilise N-15-enriched seabird-derived N as a main source of N. At a site beyond the Heron Reef Crest, macroalgae had elevated delta(15)N of 5.2parts per thousand, possibly indicating that there are locations where macroalgae access isotopically enriched aquifer-derived N. Nitrogen relations of Heron Island vegetation are compared with other reef islands and a conceptual model is presented.

Effect after introducing Bacillus thuringiensis gene on nitrogen metabolism in cotton

Bacillus thuringiensis (Bt) transgenic cotton has shown changes of vegetative and reproductive growth characteristics. The objective of this study was to investigate the physiological change of nitrogen metabolism that related closely to the growth in Bt cotton cultivars. The study Was undertaken on two 131 transgenic cotton cultivars and their parents, one conventional (Xingyang822) and recurrent parent (Sumian No. 9), the other a hybrid (Kumian No. 1) and female parent (Yumian No. 1), during the 2001 and 2002 growing seasons at the Yangzhou University Farm, Yangzhou, China. In the 2001 study, The results indicated that the Bt cotton cultivars were higher than their parents in leaf total nitrogen, free amino acid and soluble protein content, greater in NR and GPT activity, and lower in protease activity, during peak square and boll developing period. The biggest increase of total nitrogen was at peak boll period, which increased by 36.01 and 18.96% for Kumian No. I and Xingyang822, respectively. There were similar results for free amino acid and soluble protein content. The results showed further in 2002 study that NR activity increased dramatically at peak square and early boll open period, the biggest increase at early boll open period, with Kumian No. I and Xingyan,822 being 87.5 and 61.4% higher than their parent, respectively, the biggest increase of GPT activity was at peak boll period, with Kumian No. I and Xingyang822 being 39.1 and 29.1% higher than their parent, respectively. However, protease activity of Bt cultivars reduced significantly before flowering and early boll open period, the biggest decrease was before flowering period, with Kumian No. I being more than 30%, Xingyang822 being 26.5% at peak square period. Moreover, the boll total nitrogen content reduced sharply. The results suggest that the Bt cotton cultivars have higher intensity of leaf nitrogen metabolism than their parent, especially during square and boll development period. It is disadvantage for square development and earlier boll maturity under high nitrogen condition. The cultural practice should aim at reducing leaf nitrogen metabolic strength and keep the balance of vegetative and reproductive growth. (C) 2003 Elsevier B.V. All rights reserved.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division

The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.

Enzyme Electrochemistry - Biocatalysis on an electrode

Oxidoreductase enzymes catalyze single- or multi-electron reduction/oxidation reactions of small molecule inorganic or organic substrates, and they are integral to a wide variety of biological processes including respiration, energy production, biosynthesis, metabolism, and detoxification. All redox enzymes require a natural redox partner such as an electron-transfer protein ( e. g. cytochrome, ferredoxin, flavoprotein) or a small molecule cosubstrate ( e. g. NAD(P)H, dioxygen) to sustain catalysis, in effect to balance the substrate/product redox half-reaction. In principle, the natural electron-transfer partner may be replaced by an electrochemical working electrode. One of the great strengths of this approach is that the rate of catalysis ( equivalent to the observed electrochemical current) may be probed as a function of applied potential through linear sweep and cyclic voltammetry, and insight to the overall catalytic mechanism may be gained by a systematic electrochemical study coupled with theoretical analysis. In this review, the various approaches to enzyme electrochemistry will be discussed, including direct and indirect ( mediated) experiments, and a brief coverage of the theory relevant to these techniques will be presented. The importance of immobilizing enzymes on the electrode surface will be presented and the variety of ways that this may be done will be reviewed. The importance of chemical modification of the electrode surface in ensuring an environment conducive to a stable and active enzyme capable of functioning natively will be illustrated. Fundamental research into electrochemically driven enzyme catalysis has led to some remarkable practical applications. The glucose oxidase enzyme electrode is a spectacularly successful application of enzyme electrochemistry. Biosensors based on this technology are used worldwide by sufferers of diabetes to provide rapid and accurate analysis of blood glucose concentrations. Other applications of enzyme electrochemistry are in the sensing of macromolecular complexation events such as antigen - antibody binding and DNA hybridization. The review will include a selection of enzymes that have been successfully investigated by electrochemistry and, where appropriate, discuss their development towards practical biotechnological applications.

Structure of the active site of sulfite dehydrogenase from Starkeya novella

In this paper, we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized and reduced active sites of the sulfite dehydrogenase from Starkeya novella. Our results provide the first direct structural information on the active site of the oxidized form of this enzyme and confirm the conclusions derived from protein crystallography that the molybdenum coordination is analogous to that of the sulfite oxidases. The molybdenum atom of the oxidized enzyme is bound by two Mo=O ligands at 1.73 angstrom and three thiolate Mo-S ligands at 2.42 angstrom, whereas the reduced enzyme has one oxo at 1.74 angstrom, one long oxygen at 2.19 angstrom (characteristic of Mo-OH2), and three Mo-S ligands at 2.40 angstrom.

Impact of pre-existing sulfate on retention of imported chloride and nitrate in variable charge soil profiles

In variable charge soils, anion retention and accumulation through adsorption at exchange sites is a competitive process. The objectives of this study in the wet tropics of far north Queensland were to investigate (i) whether the pre-existing high sulphate in variable charge soils had any impact on the retention of chloride and nitrate, derived mostly from the applied fertilizer; and (ii) whether chloride competed with nitrate during the adsorption processes. Soil cores up to 12.5 m depth were taken from seven sites, representing four soil types, in the Johnstone River Catchment. Six of these sites had been under sugarcane (Saccharum officinarum-S) cultivation for at least 50 years and one was an undisturbed rainforest. The cores were segmented at 1.0 m depth increments, and subsamples were analysed for nitrate-N, cation (CEC)- and anion-exchange capacities (AEC), pH, exchangeable cations (Ca, Mg, K, Na), soil organic C (SOC), electrical conductivity (EC), sulphate-S, and chloride. Sulphate-S load in 1-12 m depth under cropping ranged from 9.4 to 73.9 t ha(-1) (mean= 40 t ha(-1)) compared with 74.4 t ha(-1) in the rainforest. Chloride load under cropping ranged from 1.5 to 9.6 t ha(-1) (mean= 4.9 t ha(-1)) compared to 0.9 t ha(-1) in the rainforest, and the nitrate-N load from 113 to 2760 kg ha(-1) (mean = 910 kg ha(-1)) under cropping compared to 12 kg ha(-1) in the rainforest. Regardless of the soil type, the total chloride or nitrate-N input in fertilisers was 7.5 t ha(-1), during the last 50 years. Sulphate-S distribution in soil profiles decreased with depth at >2 m, whereas bulges of chloride or nitrate-N were observed at depths >2 m. This suggests that chloride or nitrate adsorption and retention increased with decreasing sulphate dominance. Abrupt decreases in equivalent fraction of sulphate (EFSO4), at depths >2 m, were accompanied by rapid increases in equivalent fraction of chloride (EFCl), followed by nitrate (EFNO3). The stepwise regression for EFCl and EFNO3 indicated that nitrate retention was reduced by the pre-existing sulphate and imported chloride, whereas only sulphate reduced chloride adsorption. The results indicate that chloride and nitrate adsorption and retention occurred, in the order chloride>nitrate, in soils containing large amounts of sulphate under approximately similar total inputs of N- and Cl-fertilisers. (C) 2004 Elsevier B.V. All rights reserved.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo-VI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo-VI form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo-VI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Nitrate-nitrate toxicity in cattle and sheep grazing Dactyloctenium radulans (button grass) in stockyards

Hungry cattle and sheep introduced to stockyards containing a dominant or pure growth of Dactyloctenium radulans (button grass) suffered acute nitrate-nitrite toxicity in four incidents in inland Queensland between 1993 and 2001. Deaths ranged from 16 to 44%. Methaemoglobinaemia was noted at necropsies in all incidents. An aqueous humour sample from one dead steer contained 75 mg nitrate/L and from one dead sheep contained 100 mg nitrate and 50 mg nitrite/L (normal = ca 5 mg nitrate/L). Both lush and dry button grass were toxic. The nitrate content of button grass from within the stockyards ranged from 4.0 to 12.9% as potassium nitrate equivalent in dry matter and from outside the stockyards ranged from

Nodulated N-2-fixing Casuarina cunninghamiana is the sink for net N transfer from non-N-2-fixing Eucalyptus maculata via an ectomycorrhizal fungus Pisolithus sp using (NH4+)-N-15 or (NO3-)-N-15 supplied as ammonium nitrate

To determine the effects of nitrogen source on rates of net N transfer between plants connected by a common mycorrhizal network, we measured transfer of N supplied as (NH4NO3)-N-15-N-14 or (NH4NO3)-N-14-N-15 in three Casuarina/Eucalyptus treatments interconnected by a Pisolithus sp. The treatments were nonnodulated nonmycorrhizal/nonmycorrhizal; nonnodulated mycorrhizal/mycorrhizal; and nodulated mycorrhizal/mycorrhizal. Mycorrhization was 67% in Eucalyptus and 36% in Casuarina. N-2 fixation supplied 38% of the N in Casuarina. Biomass, N and N-15 contents were lowest in nonmycorrhizal plants and greatest in plants in the nodulated/mycorrhizal treatment. Nitrogen transfer was enhanced by mycorrhization and by nodulation, and was greater when N was supplied as (NH4+)-N-15 than (NO3-)-N-15. Nitrogen transfer rates were lowest in the nonmycorrhizal treatment for either N-15 source, and greatest in the nodulated, mycorrhizal treatment. Transfer was greater to Casuarina than to Eucalyptus and where ammonium rather than nitrate was the N source. Irrespective of N-15 source and of whether Casuarina or Eucalyptus was the N sink, net N transfer was low and was similar in both nonnodulated treatments. However, when Casuarina was the N sink in the nodulated, mycorrhizal treatment, net N transfer was much greater with (NH4+)-N-15 than with (NO3-)-N-15. High N demand by Casuarina resulted in greater net N transfer from the less N-demanding Eucalyptus. Net transfer of N from a non-N-2-fixing to an N-2-fixing plant may reflect the very high N demand of N-2-fixing species.

XPS study of basic aluminum sulphate and basic aluminium nitrate

Basic aluminium sulphate and nitrate crystals were prepared by forced hydrolysis of aluminium salt solution followed by precipitation with a sulphate solution or by evaporation for the basic aluminium nitrate. X-ray Photoelectron Spectroscopy (XPS) confirms the chemical composition determined by ICP-AES in earlier work. High resolution XPS scans of the individual elements allow the identification of both the central (AlO4)-Al-IV group and the 12 aluminium octahedra in the [IVAlO4AlVI(OH)(24)(H2O)(12)] building unit by two Al 2p transitions with binding energies of 73.7 and 74.2 eV in both the basic aluminium sulphate and nitrate. Four different types of oxygen atoms were identified in the basic aluminium sulphate associated with the central AlO4, OH, H2O and SO4 groups in the crystal structure with transitions at 529.4, 530.1, 530.7 and 531.8 eV, respectively. (c) 2005 Elsevier B.V. All rights reserved.

Simulated rainwater effects on anion exchange capacity and nitrate retention in Ferrosols

Nitrate (NO3) accumulations (up to 1880 kg NO3-N/ha for a 12-m profile) in the soils of the Johnstone River catchment (JRC) may pose a serious environmental threat to the Great Barrier Reef lagoon if the NO3 were released. The: leaching of artificial rainwater through repacked soil columns was investigated to determine the effect of low NO3/low ionic strength inputs on the NO3 Chemistry of the JRC profiles. Repacked soil columns were used to simulate the 11.5-m profiles, and the soil solution anion and cation concentrations were monitored at 10 points throughout the soil column. As the rainwater was applied, NO3 leached down the profile, with substantial quantities exiting the columns. Anion exchange was discounted as the major mechanism of NO3 release due to the substantial net loss of anions from the system (up to 2740 kg NO3-N/ha over the experimental period). As the soils were dominated by variable charge minerals, the effect of changing pH and ionic strength on the surface charge density was investigated in relation to the release of NO3 from the exchange. It was concluded that the equilibration of the soil solution with the low ionic strength rainwater solution resulted in a lessening of both the positive and negative surface charge. Nitrate was released into the soil solution and subsequently leached due to the lessening of the positive surface charge. Loss of NO3 from the soil profile was slow, with equivalent field release times estimated to be tens of years. Although annual release rates were high in absolute terms (up to 175 kg NO3-N/ha.year), they are only slightly greater than the current loss rates from fertilised sugarcane production (up to 50 kg NO3-N/ha.year). In addition to this, the large-scale release of NO3 from the accumulations will only occur until a new equilibrium is established with the input rainwater solution.