Hans Vilter

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

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

Extraction of proteins from material rich in anionic mucilages: partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems.

For various reasons extraction of proteins from plant material is difficult. In particular phenolic compounds and polyanionic cell-wall mucilages render conventional procedures of extraction and purification much more difficult. In this respect, aqueous polymer two-phase systems are presented as a powerful technique in extraction of vanadate-dependent bromoperoxidases from the brown macroalga Laminaria digitata, a seaweed extremely rich in mucilages. Little bromoperoxidase activity was obtained when fresh thallus material was extracted in Tris buffer. Extraction from freeze-dried and powdered material was more efficient but only satisfactory when partitioning in an aqueous polymer two-phase system was employed. Among several two-phase systems tested, one composed of poly(ethylene glycol) (PEG 1550) and potassium carbonate proved most successful (phase system-1). A rapid and efficient extraction procedure was developed with special regard for suitability in large scale processes. Staining for catalytic activity after PAGE revealed a pattern of several bromoperoxidase isoforms. Bromoperoxidases extracted in phase system-1 were fractionated into two groups of isoforms by partitioning in a second system (phase system-2) indicating that isoforms from both groups differ significantly in surface properties. Subsequently, one purification step by hydrophobic interaction chromatography was sufficient to remove residual non-peroxidase proteins as well as remaining polysaccharides from bromoperoxidases of both groups. Thus, consideration of aqueous two-phase systems as a technique for extraction and purification of plant proteins can be recommended, whenever inconveniant amounts of phenolic compounds, mucilages or pigments are present.

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.

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.

A 17O 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.

On the reactivity of bromoperoxidase I (Ascophyllum nodosum) in buffered organic media: formation of carbon bromine bonds.

Peroxidase (PO) activity of vanadate(V)-dependent bromoperoxidase (BPO) I (Ascophyllum nodosum) [VBrPO(AnI)] was retained with a half-life time of ~60 days, if stored in H2O2-incubated, morpholin-4-ethane sulfonic acid (MES)-buffered aqueous alcoholic solutions. These conditions were applied for converting bromide and, e.g., methyl pyrrole-2-carboxylate into bromopyrroles with an almost quantitative peroxide yield. δ,ε-unsaturated alcohols furnished β-bromohydrins and products of bromocyclization, i.e., tetrahydrofurans and tetrahydropyrans (70–84 % mass balance), if treated with H2O2, KBr, and VBrPO(AnI) in phosphate-buffered, CH3CN-diluted media.

K-edge X-ray absorption spectra of biomimetic oxovanadium coordination compounds

Abstract Twenty-seven coordination compounds of VO3+ and VO2+ of coordination numbers 4–7, containing biomimetic and related ligands in six different coordination geometries have been investigated, using X-ray absorption spectroscopy (XAS) in the K-edge region of vanadium. In contrast to literature reports on less variable series of complexes, no simple correlations between the energy position of the pre-edge white band (1s→3d) and the ligand electronegativities or the oxidation number of vanadium have been found. The feature of the white band, usually a composite of three transitions, and its energy (4.6–6.2 eV in V(V), 4.3–5.8 eV in V(IV) complexes) is mainly a function of geometrical distortions (vanadium-ligand bond lengths and deviation of the vanadium centre from the plane of the polyhedron). Also, there is no apparent correlation between the relative intensity of the 1s→3d transitions and the size of the coordination cage. The K edge (1s→4p) is between 20 and 23 eV with a second maximum, in most cases, at ≈35 eV. Extra features in the K edge arise for peroxo complexes (12 eV), and some octahedral and pentagonal bipyramidal complexes (17 ev).

The first crystallization of a vanadium-dependent peroxidase

Single crystals of a vanadium‐containing peroxidase from the brown alga Ascophyllum nodosum have been grown by the vapour diffusion technique using polyethylene glycol 6000 along with sodium chloride as precipitant. The crystals belong to the monoclinic space group P21. X‐ray diffraction extends to at least 2.4 Å. The cell dimensions a = 173.0, b = 164.9, c=68.5 Å, β=94.5° indicate that there are four molecules of 100 kDa per asymmetric unit, suggesting that the native enzyme might occur as a tetramer.

Detection of Vanadate-Dependent Bromoperoxidases in Protoplasts from the Brown Algae Laminaria digitata and L. saccharina

Summary Protoplasts were prepared from the brown algae Laminaria digitata (Huds.) Lamour and L. saccharina (L.) Lamour in order to investigate vanadate-dependent bromoperoxidases. In both species bromoperoxidases were present in protoplasts and quantitative differences in electrophoretic bromoperoxidase patterns were observed between small surface-cell protoplasts and vacuolated cortical-cell protoplasts as well as between stipe and thallus preparations. Bromoperoxidase patterns obtained from protoplasts and cellwall fractions differed only in the absence of electrophoretically fast migrating isoforms in protoplasts. Since extracts from entire tissues also contained these isoforms the results suggest posttranslational modification of some bromoperoxidases during or after secretion into the cell wall. Biosynthesis of the major bromoperoxidase isoform was detected in protoplasts from L. saccharina incubated with 35 S-methionine.

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.