Bea E. Krenn

A comparison of different (vanadium) bromoperoxidases; the bromoperoxidase from Corallina pilulifera is also a vanadium enzyme

Abstract The bromoperoxidase from the red seaweed Corallina pilulifera has been further characterised. The purified enzyme did not contain vanadium which was shown to be essential for enzymic activity. Upon dialysis against 0.1 M citrate/phosphate buffer (pH 3.8) containing 1 mM EDTA, the enzyme lost 77% of its brominating activity. The activity of this apo-enzyme could be reconstituted by addition of vanadate. In contrast, ferrous and ferric iron salts were unable to give reconstitution. The enzyme as isolated contained 4 mol of vanadium per mol of bromoperoxidase (molecular mmass 790 kDa) as established by atomic absorption spectrophotometry and electron paramagnetic resonance. The EPR data of the enzyme from this red alga were nearly identical to those of the vanadium-containing bromoperoxidases from the brown algae Ascophyllum nodosum and Laminaria saccharina and from the lichen Xanthoria parietina . This shows that the active sites in these enzymes are very similar. To assess any other structural similarities, the amino acid compositions and the immunological properties of these vanadium bromoperoxidases were studied. Immunotitration and Ouchterlony immunodiffusion experiments were performed using antisera raised against the bromoperoxidases from C. pilulifera and A. nodosum . Only the vanadium bromoperoxidase from the red seaweed Ceranium rubrum was partially immunologically identical to the vanadium enzyme from A. nodosum .

The bromoperoxidase from the red alga Ceramium rubrum also contains vanadium as a prosthetic group

Abstract A novel bromoperoxidase was isolated from the marine red alga Ceramium rubrum (Ceramiaceae, Rhodophyta); for the enzymic activity of this peroxidase the transition metal vanadium proved to be essential. The presence of vanadium in the enzyme preparation was established by atomic absorption spectrophotometry. Like the vanadium-containing bromoperoxidases from brown algae, this enzyme was not inhibited by cyanide or azide. This is the first report of the occurrence of a vanadium-containing bromoperoxidase in a red seaweed.

Some structural aspects of vanadium bromoperoxidase from Ascophyllum nodosum

The stability of the vanadium containing bromoperoxidase from Ascophyllum nodosum was studied. The enzyme was very resistant against chemical denaturation. Denaturation did not occur upon incubation in 4 M guanidine hydrochloride. Circular dichroism measurements showed that the secondary structure was not affected upon incubation in 4% sodium dodecyl sulphate. The sedimentation coefficient and the molecular mass, determined by ultracentrifugation were 6.96 S and 97 kDa, respectively, indicating a very compact molecule. The protein molecule contained 16 cysteine residues, all of which participated in the formation of disulfide bridges. Circular dichroism-measurements in the far ultraviolet region revealed that the protein consisted of a large amount of alpha-helix (74%), and no beta-pleated sheet. The dissociation constant of the apoprotein vanadium-complex was 55 nM (at pH 8.5), and rapidly increased at lower pH. The data suggest that the protonation of a group with a pKa higher than 8.5 prevents the binding of vanadate. Structural analogues of vanadate (phosphate and arsenate) were competitive inhibitors with respect to the reconstitution of the bromoperoxidase. The inhibition constants were 60 and 120 microM for phosphate and arsenate, respectively. The binding of hydrogen peroxide to the enzyme was visualized by optical spectroscopy. Upon addition of H2O2 the optical absorption spectrum showed a small, but significant, decrease in absorption in the 315 nm region, which was restored upon addition of bromide, or by allowing the solution to stand for several hours. These changes are ascribed to the formation of a stable enzyme-peroxo-intermediate, in line with a previous analysis of the steady-state kinetics.

Purification and some characteristics of a non-haem bromoperoxidase from Streptomyces aureofaciens.

A bromoperoxidase was isolated from the chlortetracycline-producing actinomycete, Streptomyces aureofaciens. This enzyme catalysed bromination and iodination, but surprisingly did not catalyse chlorination. The enzyme had an acidic pH optimum (pH 4.3) and the isoelectric point was 3.5. The Km for bromide was 20 mM and the Km for H2O2 was as high as 8 mM. The bromoperoxidase did not contain haem, since it was not inhibited by azide or cyanide. Excess bromide or chloride had no effect on its brominating activity; however, fluoride strongly inhibited the bromoperoxidase (Ki = 20 microM). On the basis of gel electrophoresis in the absence and presence of sodium dodecyl sulphate, the molecular mass of the enzyme was 65 kDa and it consisted of two subunits of 32 kDa each. The bromoperoxidase was remarkably thermostable.

Vanadium ― an element involved in the biosynthesis of halogenated compounds and nitrogen fixation

In nature, and in particular in the marine environment, a wide variety of halometabolites is found. Marine algae, for example, are a rich source of bromometabolites [l]. Some of the algae, such as the brown seaweeds (Phaeophyta) Ascophyilum nodosum and Fucus vesiculosus, contribute enormous quantities of organo-halo species to our ecosystem and form an important source of bromine-containing material released to the atmosphere [2]. The role of these halogenated compounds is not always clear. Some appear to participate in biological defence mechanisms, since they show antimicrobial or antifeeding properties, others may act as chemical messengers in communication systems. A number of enzymes involved in the biosynthesis of these compounds have been purified and characterised, and it was up to now generally accepted that peroxidases containing haem are involved in the halogenation reaction. The mechanism by which halogenation occurs is still open to debate (discussed in the book by Neidleman and Geigert [ 11). Some authors suggest that the haloperoxidases generate free hypohalous acid which will react aspecifically with a broad range of nucleophilic acceptors, whereas

Patterned electroplating of micrometer scale magnetic structures on glass substrates

This paper has developed a new method of micro patterned electroplating that enables the fabrication of micrometer scale magnetic structures on glass substrates. In contrast to other methods, the process as developed here leaves the surrounding substrate area untouched: that is there is no seed layer present except underneath the magnetic structures. Patterned cobalt layers of thicknesses up to 8 /spl mu/m were fabricated exhibiting high saturation (1.5 T) and low coercive force. These layers have been used to create micrometer scale magnetic poles (magnetic tweezers) for biological and biophysical applications, where a clean untouched glass substrate in the sample area is most important. The technique, however, can be used in any situation where a residual seed layer on the surrounding area is unwanted, and afterwards removal of this seed layer is difficult or impossible.

Co-reconstitution of the H+-ATP synthase and cytochrome b-563c-554 complex from a thermophilic cyanobacterium: High ATP yield and mutual effects on the enzymatic activities

Abstract The H+-ATP synthase and the cytochrome b-563 c-554 (Qbc) complex were selectively solubilized and isolated from the thylakoid membranes of the thermophilic cyanobacterium Synechococcus 6716. The enzymes were co-reconstituted into proteoliposomes with the native cyanobacterial lipids by detergent dialysis. After (co-)reconstitution of the protein complexes in variable stoichiometries, the different enzymatic activities were analyzed. Optimization of the functionally coupled enzymes led to a compromise for the weight ratios of 20 mg lipid and 0.5 mg of both complexes per ml of reconstitution medium. The average diameter (around 300 nm) and zeta potential of these proteoliposomes were determined. The oxidoreductase activity of reconstituted Qbc complex was found to be stimulated by the presence of co-reconstituted (inactive) ATP synthase. Conversely, ATP hydrolysis was inhibited or stimulated by the protonmotive force generated by the Qbc complex, co-reconstituted in the right-side out or inside-out orientation, respectively. In proteoliposomes containing trapped horse-heart cytochrome c the proton movement induced by the Qbc complex resulted in an ATP production of about 1 μmol ATP per mg of ATP synthase within 1 min. The mutual stimulation and high ATP synthesis activity may indicate improved incorporation and/or some kind of functional association of these proteins in the membrane, although as yet aspecific protein-stabilizing effects cannot be excluded.

Marine Vanadium-Dependent Haloperoxidases, Their Isolation, Characterization, and Application

Vanadium-dependent haloperoxidases in seaweeds, cyanobacteria, fungi, and possibly phytoplankton play an important role in the release of halogenated volatile compounds in the environment. These halocarbons have effects on atmospheric chemistry since they cause ozone depletion. In this chapter, a survey is given of the different sources of these enzymes, some of their properties, the various methods to isolate them, and the bottlenecks in purification. The assays to detect and quantify haloperoxidase activity are described as well as their kinetic properties. Several practical tips and pitfalls are given which have not yet been published explicitly. Recent developments in research on structure and function of these enzymes are reviewed. Finally, the application of vanadium-dependent haloperoxidases in the biosynthesis of brominated and other compounds is discussed.