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.

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.

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.

Crystallization and preliminary X-ray analysis of a vanadium-dependent peroxidase from Ascophyllum nodosum.

Peroxidase from the brown alga Ascophyllum nodosum, a non-heme vanadium-dependent haloperoxidase, has been purified to homogeneity and crystallized from ammonium sulfate solutions in a form suitable for X-ray diffraction analysis. The crystals have been grown by the vapour-diffusion technique using the sitting-drop method. X-ray diffraction studies show that the crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 114.3 and c = 276.0 A. The crystals diffract to at least 2.4 A resolution.

High-resolution XANES studies on vanadium-containing haloperoxidase: pH-dependence and substrate binding.

High-resolution X-ray absorption vanadium K-edge spectra were recorded for samples of vanadium-containing bromoperoxidase from the brown alga, Ascophyllum nodosum, at pH 9, 7, 5 and 4, as well as for enzyme samples containing the substrates, hydrogen peroxide and bromide. The well-resolved features of the XANES spectra are discussed. The pH-dependence of the structure of the active site has been studied revealing no significant change of the absorption features. We were able to detect an interaction of H2O2 with the vanadium site of the bromoperoxidase using XAS spectroscopy, whereas addition of bromide causes no energy shift of the XANES spectrum. The possible role of vanadium during the enzymatic reaction is discussed on the basis of our results.

An improved method for the purification of lignin peroxidases from Phanerochaete chrysosporium INA-12: properties of two major isoforms.

1. Phanerochaete chrysosporium INA-12 secretes several lignin peroxidase isoenzymes. This paper reports an improved procedure for the purification of the different isoforms compared to those previously described. 2. Lignin peroxidases are first concentrated and prefractionated on fast-flow ion-exchangers which avoid concentration by ultrafiltration and dialysis. 3. Further purification is achieved by hydrophobic interaction chromatography and anion-exchange FPLC. 4. Two major forms were purified to homogeneity. Kinetic measurements and protein characterization (isoelectric points, phosphate content) suggest that they are similar to those produced by P. chrysosporium BKM strain.

(Model) studies on vanadate-dependent bromo/iodoperoxidase from Ascophyllum nodosum. VO2+ is not incorporated into the active site.

Vanadate-dependent peroxidase A.n.I, the main isoenzyme (M(r) = 100 kDa) from the seaweed, Ascophyllum nodosum, contains 2 V per enzyme molecule (as shown by ICP-MS metal analysis) after complete reconstitution with vanadate (V), possibly distributed in a 1:1 ratio between the surface and active site. VO2+ is only weakly associated to the surface of A.n.I. There is no transport channel for VO2+. The EPR spectrum of the reduced holoenzyme is anisotropic (axial) already at room temperature, with EPR parameters similar to those of VO2+ complexes of small model peptides such as Ala-His, Gly-Tyr, Gly-Ser, Gly-Glu, Ser-Gly and Phe-Glu. The complex formation between Ala-His and H2VO4- in water has also been investigated (by 51V NMR); the formation constant at pH 7.2 amounts to 266(28) M-1.

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.

Native bromoperoxidases do not bind to nitrocellulose: use of DEAE-cellulose as an alternative in blotting.

Bromoperoxidases were investigated by protein blotting after polyacrylamide gel electrophoresis. While the denatured proteins bound to nitrocellulose, the native enzymes did not. Instead, they could be transferred successfully to DEAE-cellulose. Procedures for immunostaining and glycoprotein detection with concanavalin A on DEAE-cellulose are described. The results indicate that binding of native proteins to nitrocellulose can not necessarily be assumed. DEAE-cellulose is suitable both to investigate this phenomenon or as a substitute for nitrocellulose in blotting of native proteins.

Aqueous two-phase extraction of plant enzymes from sources containing large amounts of tannins and anionic mucilages.

The isolation of plant enzymes is frequently hampered by the presence of phenolic compounds, pigments and mucilages. Recently, extraction procedures based on aqueous two-phase systems were used to overcome these problems. Two-phase systems have a great advantage in respect to yield, product purity and processing time. Two-phase systems may open many new avenues in research as well as for application of enzymes from plant material, especially making available enzymes of sources avoided till now, due to the difficulties to work with.

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.