Vanadium K-edge absorption spectrum of bromoperoxidase from Ascophyllum nodosum

With synchrotron radiation from the Bonn 2.5 GeV synchrotron, high-resolution absorption spectra have been measured at the vanadium K-edge of bromoperoxidase from the marine brown alga Ascophyllum nodosum and several model compounds. The near-edge structure (XANES) of these spectra was used to determine the charge state and the coordination geometry around the vanadium atom. For the active enzyme a coordination charge of 2.7 was found which is compatible with a formal valence of +5, assuming coordination by atoms with a high electronegativity such as oxygen or nitrogen. For the reduced enzyme the coordination charge value of 2.15 indicates the reduction of the valency by 1 unit. Our results suggest that the coordination sphere of the vanadium atom in the native enzyme consists of at least seven oxygen atoms in a distorted octahedral environment with an average bond length of about 2 A. Through the reduction process, the coordination sphere of the vanadium atom changes with a simultaneous decrease of the coordination cage. These results agree with those deduced from previous EPR and 51V-NMR measurements.     

Vanadium K-edge X-ray absorption spectroscopy of bromoperoxidase from Ascophyllum nodosum

Bromoperoxidase from Ascophyllum nodusum was the first vanadium-containing enzyme to be isolated. X-ray absorption spectra have now been collected in order to investigate the coordination of vanadium in the native, native plus bromide, native plus hydrogen peroxide, and dithionite-reduced forms of the enzyme. The edge and X-ray absorption near-edge structures show that, in the four samples studied, it is only on reduction of the native enzyme that the metal site is substantially altered. In addition, these data are consistent with the presence of vanadium(IV) in the reduced enzyme and vanadium(V) in the other samples. Extended X-ray absorption fine structure data confirm that there are structural changes at the metal site on reduction of the native enzyme, notably a lengthening of the average inner-shell distance, and the presence of terminal oxygen together with histidine and oxygen-donating residues.     

Bromine K-edge EXAFS studies of bromide binding to bromoperoxidase from Ascophyllum nodosum.

Bromine K-edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris-buffered) bromide and vanadium model compounds containing Br-V, Br-C(aliphatic) and Br-C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine.     

Molecular cloning,structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum

The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k_cat = 153 s⁻¹, 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata.     

A fucoidan fraction from Ascophyllum nodosum.

A fucoidan fraction was purified from the brown alga Ascophyllum nodosum. The polysaccharide contained L-fucose and sulfate as the only constituents. Combination of methylation analysis, Smith degradation, FTIR and NMR spectroscopy on the native and the de-sulfated polymers demonstrated that the fucoidan consisted of a highly branched core region with primarily alpha-(1-->3)-linked fucosyl residues and a few alpha-(1-->4) linkages. Branch points were at position 2 of the -->3-linked internal residues. The side chains consisted of single and multi-unit fucosyl residues. The combined analytical data suggested also a complex sulfation pattern with substitution principally at position 2 and/or position 4. Such diversity in the structural features of this fucoidan may be of importance for its various biological properties.     

Peroxidases from phaeophyceae: A vanadium(V)-dependent peroxidase from Ascophyllum nodosum

A peroxidase isolated from Ascophyllum nodosum was completely inactivated by dialysis in pH 3.8 citrate-phosphate buffer containing EDTA. The enzym     

Bromoperoxidase activity and vanadium level of the brown alga Ascophyllum nodosum

Vanadium-dependent peroxidase activity in extracts of Ascophyllum nodosum growing in the intertidal region close to Roscoff/France, and algal vanadium levels, followed approximately similar seasonal variation, as deduced from a study lasting from April 2005 to March 2006. High peroxidase (PO) activity was found in extracts obtained from algae collected in between midwinter to spring [∼100-190 U per g dry mass (dm), triiodide assay] with a maximum in April. Periods of reduced PO activity lasted from summer to early winter (∼50-90 U per g dm). High vanadium levels (1.5-2.2 mg kg⁻¹ dm) were found in algae collected from midwinter to spring, whereas reduced levels (0.6-1.4 mg kg⁻¹ dm) were found in summer to early winter.     

A (17)O NMR study of peroxide binding to the active centre of bromoperoxidase from Ascophyllum nodosum

The (17)O NMR of bromoperoxidase in Tris buffer at pH 8 treated with (17)O-enriched H2O2 reveals direct binding of peroxide to active site vanadium both in the symmetric and asymmetric modes, the latter possibly due to hydroperoxide. In addition, non-active site HVO2(O2)2(2-) is detected. The results are counter-checked with NMR data on peroxovanadium model compounds.     

Characterization of vanadium bromoperoxidase from Macrocystis and Fucus: reactivity of vanadium bromoperoxidase toward acyl and alkyl peroxides and bromination of amines

Vanadium bromoperoxidase (V-BrPO) has been isolated and purified from the marine brown algae Fucus distichus and Macrocystis pyrifera. V-BrPO catalyzes the oxidation of bromide by hydrogen peroxide, resulting in the bromination of certain organic acceptors or the formation of dioxygen. V-BrPO from F. distichus and M. pyrifera have subunit molecular weights of 65,000 and 74,000, respectively, and specific activities of 1580 units/mg (pH 6.5) and 1730 units/mg (pH 6) for the bromination of monochlorodimedone, respectively. As isolated, the enzymes contain a substoichiometric vanadium/subunit ratio; the vanadium content and specific activity are increased by addition of vanadate. V-BrPO (F. distichus, M. pyrifera, and Ascophyllum nodosum) also catalyzes the oxidation of bromide using peracetic acid. In the absence of an organic acceptor, a mixture of oxidized bromine species (e.g., hypobromous acid, bromine, and tribromide) is formed. Bromamine derivatives are formed from the corresponding amines, while 5-bromocytosine is formed from cytosine. In all cases, the rate of the V-BrPO-catalyzed reaction is much faster than that of the uncatalyzed oxidation of bromide by peracetic acid, at pH 8.5, 1 mM bromide, and 2 mM peracetic acid. In contrast to hydrogen peroxide, V-BrPO does not catalyze formation of dioxygen from peracetic acid in either the presence or absence of bromide. V-BrPO also uses phenylperacetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid to catalyze the oxidation of bromide; dioxygen is not formed with these peracids. V-BrPO does not catalyze bromide oxidation or dioxygen formation with the alkyl peroxides ethyl hydroperoxide, tert-butyl hydroperoxide, and cuminyl hydroperoxide.     

Reactivation of vanadium bromoperoxidase; inhibition by metallofluoric compounds.

The effect of phosphate analogs (pyrophosphate, aluminofluoride and beryllofluoride complexes) on the reactivation of apobromoperoxidase by vanadate was studied. P2O7(4-) inhibited the reactivation in the millimolar range. Of the different aluminofluoride complexes, only AlF4- was inhibitory. In addition, BeF4(2-) also appeared to bind with high affinity to the apobromoperoxidase, thus inhibiting the reactivation very strongly. The inhibition observed supports a mechanism in which the fluorometallic complexes act as analogs of vanadate and bind accordingly to the apobromoperoxidase.     

The bromoperoxidase from the lichen Xanthoria parietina is a novel vanadium enzyme.

A novel bromoperoxidase was isolated from the lichen Xanthoria parietina. The enzyme contained vanadium, which is essential for enzymic activity. Under denaturating conditions the preparation showed a single protein band with an Mr of 65,000. Thermal-denaturation studies showed that this bromoperoxidase could tolerate high temperatures. The affinity of the enzyme for its substrate bromide is high; the Km for bromide was 29 microM. Excess halides (50 mM) inhibited enzymic activity considerably.     

Electrospray ionization mass spectrometry of oligosaccharides derived from fucoidan of Ascophyllum nodosum

Algal fucoidan is a complex sulfated polysaccharide whose structural characterization requires powerful spectroscopic methodologies. While most of the structural investigations reported so far have been performed using NMR as the main spectroscopic method, we report herein data obtained by negative electrospray ionization mass spectrometry. MS analysis has been carried out on oligosaccharides obtained by partial hydrolysis of fucoidan from the brown algae Ascophyllum nodosum. Oligosaccharide mixtures were fractionated by size exclusion chromatography, which allowed the analysis of oligomers ranging from monosaccharide to pentasaccharide. Monosaccharides were detected as monosulfated as well as disulfated forms. Besides, part of the oligosaccharides exhibited a high content of sulfate, evidencing that fucoidan contains disulfated fucosyl units. Fragmentation experiments yielded characteristic fragment ions indicating that the fucose units are mainly 2-O-sulfated. This study demonstrates that highly sulfated oligosaccharides from fucoidan can be analyzed by ESIMS which gives additional information about the structure of this highly complex polysaccharide.     

X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 A resolution.

The homo-dimeric structure of a vanadium-dependent haloperoxidase (V-BPO) from the brown alga Ascophyllum nodosum (EC 1.1.11.X) has been solved by single isomorphous replacement anomalous scattering (SIRAS) X-ray crystallography at 2.0 A resolution (PDB accession code 1QI9), using two heavy-atom datasets of a tungstate derivative measured at two different wavelengths. The protein sequence (SwissProt entry code P81701) of V-BPO was established by combining results from protein and DNA sequencing, and electron density interpretation. The enzyme has nearly an all-helical structure, with two four-helix bundles and only three small beta-sheets. The holoenzyme contains trigonal-bipyramidal coordinated vanadium atoms at its two active centres. Structural similarity to the only other structurally characterized vanadium-dependent chloroperoxidase (V-CPO) from Curvularia inaequalis exists in the vicinity of the active site and to a lesser extent in the central four-helix bundle. Despite the low sequence and structural similarity between V-BPO and V-CPO, the vanadium binding centres are highly conserved on the N-terminal side of an alpha-helix and include the proposed catalytic histidine residue (His418(V-BPO)/His404(V-CPO)). The V-BPO structure contains, in addition, a second histidine near the active site (His411(V-BPO)), which can alter the redox potential of the catalytically active VO2-O2 species by protonation/deprotonation reactions. Specific binding sites for the organic substrates, like indoles and monochlordimedone, or for halide ions are not visible in the V-BPO structure. A reaction mechanism for the enzymatic oxidation of halides is discussed, based on the present structural, spectroscopic and biochemical knowledge of vanadium-dependent haloperoxidases, explaining the observed enzymatic differences between both enzymes.     

Efficient purification with high recovery of vanadium bromoperoxidase from Corallina officinalis

A novel, simple and highly efficient process for purifying vanadium bromoperoxidase from Corallina officinalis is reported. The key innovation is adding 0.5 mM sodium orthovanadate to the crude cell extract followed by heating at 70°C for 2 h, by which a 5.4-fold purification with a 100% activity recovery was achieved. Combining the heat treatment with ammonium sulfate precipitation and DEAE-52 column chromatography, the overall yield was 84%, 3.8 times greater than the highest yield previously reported. Finally, a specific activity of 310 U/mg, a 27-fold purification of the crude enzyme solution was produced.     

Antidiabetic properties of polysaccharide- and polyphenolic-enriched fractions from the brown seaweed Ascophyllum nodosum

We screened seaweed species from Atlantic Canada for antidiabetic activity by testing extracts for alpha-glucosidase inhibitory effect and glucose uptake stimulatory activity. An aqueous ethanolic extract of Ascophyllum nodosum was found to be active in both assays, inhibiting rat intestinal alpha-glucosidase (IC50 = 77 microg/mL) and stimulating basal glucose uptake into 3T3-L1 adipocytes during a 20-minute incubation by about 3-fold (at 400 microg/mL extract). Bioassay-guided fractionation of the A. nodosum extract showed that alpha-glucosidase inhibition was associated with polyphenolic components in the extract. These polyphenolics, along with other constituents appeared to be responsible for the stimulatory activity on glucose uptake. However, attempts to further concentrate this activity through fractionation techniques were unsuccessful. A crude polyphenol extract (PPE), an enriched polyphenolic fraction (PPE-F1) and a polysaccharide extract (PSE) were prepared from commercial A. nodosum powder and administered to streptozotocin-diabetic mice for up to 4-weeks by daily gavage at 200 mg/kg body mass. PPE and PPE-F1 improved fasting serum glucose level in diabetic mice; however, the effect was only statistically significant at day 14. In addition, PPE-F1 was shown to blunt the rise in blood glucose after an oral sucrose tolerance test in diabetic mice. Mice treated with PPE and PPE-F1 had decreased blood total cholesterol and glycated serum protein levels compared with untreated diabetic mice, whereas PPE also normalized the reduction in liver glycogen level that occurred in diabetic animals. All 3 A. nodosum preparations improved blood antioxidant capacity.     

Electron microscopy of human erythrocytes agglutinated by lectin from Codium fragile ssp. tomentosoides and pseudo-haemagglutinin from Ascophyllum nodosum

Human erythrocytes were exposed to extracts of Codium fragile ssp. tomentosoides, Ascophyllum nodosum, Dolichos biflorus seeds and a solution of purified polyphenols from Ascophyllum nodosum. In each case the erythrocytes appeared to agglutinate. Agglutinates were examined by scanning electron microscopy. Photomicrographs of the erythrocytes exposed to lectin-containing extracts of C. fragile and D. biflorus showed the typical appearance of agglutinated cells. Photomicrographs of the erythrocytes exposed to the crude extract and purified polyphenols from A. nodosum were similar to each other, but quite different in appearance to erythrocytes agglutinated by the lectins from C. fragile and D. biflorus. This evidence adds further support to the contention that lectin presence recorded in brown algal extracts is largely due to ‘pseudoagglutination’ produced by polyphenols contained in these extracts.     

Reactivity of recombinant and mutant vanadium bromoperoxidase from the red alga Corallina officinalis

Vanadium bromoperoxidase (VBPO) from the marine red alga Corallina officinalis has been cloned and heterologously expressed in Esherichia coli. The sequence for the full-length cDNA of VBPO from C. officinalis is reported. Steady state kinetic analyses of monochlorodimedone bromination reveals the recombinant enzyme behaves similarly to native VBPO from the alga. The kinetic parameters (K(m)(Br-)=1.2 mM, K(m)(H(2)O(2))=17.0 microM) at the optimal pH 6.5 for recombinant VBPO are similar to reported values for enzyme purified from the alga. The first site-directed mutagenesis experiment on VBPO is reported. Mutation of a conserved active site histidine residue to alanine (H480A) results in the loss of the ability to efficiently oxidize bromide, but retains the ability to oxidize iodide. Kinetic parameters (K(m)(I-)=33 mM, K(m)(H(2)O(2))=200 microM) for iodoperoxidase activity were determined for mutant H480A. The presence of conserved consensus sequences for the active sites of VBPO from marine sources shows its usefulness in obtaining recombinant forms of VBPO. Furthermore, mutagenesis of the conserved extra-histidine residue shows the importance of this residue in the oxidation of halides by hydrogen peroxide.     

Mechanistic investigations of the novel non-heme vanadium bromoperoxidases. Evidence for singlet oxygen production

Three newly discovered non-heme bromoperoxidases isolated from marine algae were found to catalyze the production of singlet oxygen in reactions composed of the bromoperoxidase, hydrogen peroxide, and bromide. The bromoperoxidases studied were vanadium bromoperoxidase (V-BrPO) from Ascophyllum nodosum, native non-heme bromoperoxidase from Corallina vancouveriensis (which contains vanadium and iron), and the vanadium-reconstituted bromoperoxidase derivative from C. vancouveriensis. These enzyme systems generated near infrared emission, characteristic of singlet oxygen. The emission had a peak intensity near 1268 nm, was greatly increased in 2H2O-containing buffers, and was greatly decreased by the singlet oxygen quenchers, histidine and azide. The yield of singlet oxygen was approximately 80% of the theoretical yield. A unique feature of the non-heme bromoperoxidases distinct from the iron heme haloperoxidases, was the remarkable stability of the non-heme enzymes in the presence of singlet oxygen and oxidized bromine species. V-BrPO turned over multiple aliquots of 2 mM hydrogen peroxide without losing efficiency. In contrast, iron heme lactoperoxidase was completely inactivated after turnover of the first aliquot of 2 mM hydrogen peroxide, and iron heme chloroperoxidase was 50% deactivated. The profile of singlet oxygen formation by V-BrPO and the near stoichiometric yield of singlet oxygen suggest that the mechanism of singlet oxygen formation is the same as the mechanism of dioxygen formation determined by oxygen probe measurements.     

Mechanism of dioxygen formation catalyzed by vanadium bromoperoxidase. Steady state kinetic analysis and comparison to the mechanism of bromination

The steady state kinetic mechanism of the bromide-assisted disproportionation of hydrogen peroxide, forming dioxygen, catalyzed by vanadium bromoperoxidase has been investigated and compared to the mechanism of monochlorodimedone (MCD) bromination under conditions of 0.0125-6 mM H2O2, 1-500 mM Br-, and pH 4.55-6.52. Under these conditions, 50 microM MCD was sufficient to inhibit at least 90% of the dioxygen formation during MCD bromination. The rate data is consistent with a substrate-inhibited Bi Bi Ping Pong mechanism, in which the substrate bromide, is also an inhibitor at pH 4.55 and 5.25, but not at pH 5.91 and 6.52. The kinetic parameter KmBr, KmH2O2, KisBr, and KiiBr determined for the reactions of bromide-assisted disproportionation fo hydrogen peroxide and MCD bromination are similar, indicating that the mechanisms of both reactions occur via the formation of a common intermediate, the formation of which is rate-limiting. Fluoride is a competitive inhibitor with respect to hydrogen peroxide in both reactions at pH 6.5. At high concentrations of hydrogen peroxide, the bromide-assisted disproportionation of hydrogen peroxide occurs during the bromination of MCD. The sum of the rates of MCD bromination and dioxygen formation during MCD bromination is equal to the rate of dioxygen formation in the absence of MCD. The apportionment of the reaction through the MCD bromination and dioxygen formation pathways depends on pH, with much lower hydrogen peroxide concentrations causing significant dioxygen formation at higher pH.