Environmental Control of Vanadium Haloperoxidases and Halocarbon Emissions in Macroalgae

Vanadium-dependent haloperoxidases (V-HPO), able to catalyze the reaction of halide ions (Cl^?, Br^?, I^-) with hydrogen peroxide, have a great influence on the production of halocarbons, which in turn are involved in atmospheric ozone destruction and global warming. The production of these haloperoxidases in macroalgae is influenced by changes in the surrounding environment. The first reported vanadium bromoperoxidase was discovered 40 years ago in the brown alga Ascophyllum nodosum. Since that discovery, more studies have been conducted on the structure and mechanism of the enzyme, mainly focused on three types of V-HPO, the chloro- and bromoperoxidases and, more recently, the iodoperoxidase. Since aspects of environmental regulation of haloperoxidases are less well known, the present paper will focus on reviewing the factors which influence the production of these enzymes in macroalgae, particularly their interactions with reactive oxygen species (ROS).     

Vanadium haloperoxidases from brown algae of the Laminariaceae family

Vanadium haloperoxidases were extracted, purified and characterized from three different species of Laminariaceae--Laminaria saccharina (Linné) Lamouroux, Laminaria hyperborea (Gunner) Foslie and Laminaria ochroleuca de la Pylaie. Two different forms of the vanadium haloperoxidases were purified from L. saccharina and L. hyperborea and one form from L. ochroleuca species. Reconstitution experiments in the presence of several metal ions showed that only vanadium(V) completely restored the enzymes activity. The stability of some enzymes in mixtures of buffer solution and several organic solvents such as acetone, ethanol, methanol and 1-propanol was noteworthy; for instance, after 30 days at least 40% of the initial activity for some isoforms remained in mixtures of 3:1 buffer solution/organic solvent. The enzymes were also moderately thermostable, keeping full activity up to 40 degrees C. Some preliminary steady-state kinetic studies were performed and apparent Michaelis-Menten kinetic parameters were determined for the substrates iodide and hydrogen peroxide. Histochemical studies were also performed in fresh tissue sections from stipe and blade of L. hyperborea and L. saccharina, showing that haloperoxidase activity was concentrated in the external cortex near the cuticle, although some activity was also observed in the inner cortical region.     

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K _m ^I– =2.5 mM; k _cat ^I– =462 s^–1) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K _m ^I– =18.1 mM; k _cat ^I– =38 s^–1). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.The nucleotide sequence reported in this paper has been submitted to the EBI Data Bank with accession no. AJ619804.     

Bromoperoxidase activity of vanadate-substituted acid phosphatases from Shigella flexneri and Salmonella enterica ser. typhimurium.

Vanadium haloperoxidases and the bacterial class A nonspecific acid phosphatases have a conserved active site. It is shown that vanadate-substituted recombinant acid phosphatase from Shigella flexneri (PhoN-Sf) and Salmonella enterica ser. typhimurium (PhoN-Se) in the presence of H2O2 are able to oxidize bromide to hypobromous acid. Vanadate is essential for this activity. The kinetic parameters for the artificial bromoperoxidases have been determined. The Km value for H2O2 is about the same as that for the vanadium bromoperoxidases from the seaweed Ascophyllum nodosum. However, the Km value for Br- is about 10-20 times higher, and the turnover values of about 3.4 min-1 and 33 min-1 for PhoN-Sf and PhoN-Se, respectively, are much slower, than those of the native bromoperoxidase. Thus, despite the striking similarity in the active-site structures of the vanadium haloperoxidases and the acid phosphatase, the turnover frequency is low, and clearly the active site of acid phosphatases is not optimized for haloperoxidase activity. Like the native vanadium bromoperoxidase, the vanadate-substituted PhoN-Sf and PhoN-Se catalyse the enantioselective sulfoxidation of thioanisole.     

Enzymology and Genetics of Halogenating Enzymes from Bacteria

This paper discusses the properties of bacterial haem-containing and non-haem haloperoxidases, their involvement in the biosynthesis of halometabolites and their use in bioconversion. The very low peroxidase activity of bacterial non-haem haloperoxidases, their stability at high temperature and over a wide pH-range makes them particularly suited for use in the bromination of organic compounds. The chloroperoxidase from Pseudomonas pyrrocinia is the only haloperoxidase showing substrate specificity and regioselectivity. The genes of one chloro- and one bromoperoxidase could be cloned. The corresponding enzymes can now be produced in large amounts and at low costs.     

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.     

A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities

The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H₂O₂/I⁻ catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (K_m and k_cat) of bromide and iodide for vanadium bromoperoxidase from Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.     

The brown alga Ascophyllum nodosum contains two different vanadium bromoperoxidases

Haloperoxidases have been detected in a variety of organisms, including bacteria, fungi, algae, and mammals. Mammalian haloperoxidases are known to be directly involved in the oxidative destruction of microorganisms. The algal bromoperoxidases are probably involved in the biosynthesis of bromometabolites, most of which show considerable bactericidal activity. From the brown seaweed Ascophyllum nodosum (order, Fucales) two different bromoperoxidases have been isolated, which both contain vanadium as an essential element for enzymic activity. The location of these two enzymes, determined by activity staining of cross-sections of algal parts, was different. Bromoperoxidase I (which has been described before) was located inside the thallus, particularly around the conceptacles, whereas bromoperoxidase II was present at the thallus surface of the alga. The molecular masses of these bromoperoxidases as judged from sodium dodecyl sulfate-gel electrophoresis were 97 and 106 kDa, respectively. Some of the enzymatic properties (pH optimum and Km for bromide) of the two enzymes were slightly different, whereas the amino acid compositions were more or less equal. The isoelectric point of the two proteins was the same, namely 5.0. On sodium dodecyl sulfate-polyacrylamide gels both enzymes could be stained with periodic acid Schiff's reagent, so both are glycoproteins. Since only bromoperoxidase II could be bound to a concanavalin A-Sepharose column, these enzymes contain different carbohydrates. Both enzymes display a considerable thermostability. However, the chemical stability of the two bromoperoxidases differed. Bromoperoxidase II could also be inactivated by dialysis at low pH and reactivation was only possible with the transition metal vanadium and not with other metal ions. The presence of vanadium in this enzyme could be established with atomic absorption spectrophotometry and electron paramagnetic resonance. The EPR signals of both bromoperoxidases, which were observed after reduction with sodium dithionite, were similar: only minor differences were observed in the hyperfine coupling. In immunoblotting experiments these two bromoperoxidases were found to cross-react, so they have common antigenic determinants.     

Laboratory-evolved vanadium chloroperoxidase exhibits 100-fold higher halogenating activity at alkaline pH: catalytic effects from first and second coordination sphere mutations

Directed evolution was performed on vanadium chloroperoxidase from the fungus Curvularia inaequalis to increase its brominating activity at a mildly alkaline pH for industrial and synthetic applications and to further understand its mechanism. After successful expression of the enzyme in Escherichia coli, two rounds of screening and selection, saturation mutagenesis of a "hot spot," and rational recombination, a triple mutant (P395D/L241V/T343A) was obtained that showed a 100-fold increase in activity at pH 8 (k(cat) = 100 s(-1)). The increased K(m) values for Br(-) (3.1 mm) and H(2)O(2) (16 microm) are smaller than those found for vanadium bromoperoxidases that are reasonably active at this pH. In addition the brominating activity at pH 5 was increased by a factor of 6 (k(cat) = 575 s(-1)), and the chlorinating activity at pH 5 was increased by a factor of 2 (k(cat) = 36 s(-1)), yielding the "best" vanadium haloperoxidase known thus far. The mutations are in the first and second coordination sphere of the vanadate cofactor, and the catalytic effects suggest that fine tuning of residues Lys-353 and Phe-397, along with addition of negative charge or removal of positive charge near one of the vanadate oxygens, is very important. Lys-353 and Phe-397 were previously assigned to be essential in peroxide activation and halide binding. Analysis of the catalytic parameters of the mutant vanadium bromoperoxidase from the seaweed Ascophyllum nodosum also adds fuel to the discussion regarding factors governing the halide specificity of vanadium haloperoxidases. This study presents the first example of directed evolution of a vanadium enzyme.     

The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.     

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.     

Purification, characterization and comparison of two non-haem bromoperoxidases from Streptomyces aureofaciens ATCC 10762.

Two non-haem bromoperoxidases (BPO 1 and BPO 2) were purified from the 7-chlorotetracycline-producing strain Streptomyces aureofaciens ATCC 10762. Both enzymes showed azide-insensitive brominating activity, and bromide-dependent peroxidase activity. BPO 1 was a dimer (Mr 65,000) with subunits of identical size (Mr 31,000). The pI was estimated to be 4.5. The enzyme did not cross-react with antibodies raised against the non-haem bromoperoxidase (Mr 90,000) from S. aureofaciens Tü24, a strain that also produces 7-chlorotetracycline. The Mr of BPO 2 was estimated to be 90,000. The enzyme had three identical subunits (Mr 31,000), and its isoelectric point was 3.5, identical with that of the bromoperoxidase from S. aureofaciens Tü24. Moreover, BPO 2 was immunologically identical with the bromoperoxidase from S. aureofaciens Tü24, although both it and BPO 1 could be distinguished electrophoretically from the latter bromoperoxidase.     

Purification and partial characterization of multiple bromoperoxidases from Streptomyces griseus

The presence of multiple bromoperoxidases in extracts of Streptomyces griseus Tü 6 was detected. The enzyme pattern varied with the age of the culture. A haem-type bromoperoxidase (BPO 2) was always present. Additionally three nonhaem-type bromoperoxidases (BPO 1a, 1b and 3) were detected and purified to homogeneity. The Mr of non-denatured BPO 1a was 70,000 +/- 10,000 and those of BPO 1b and 3 were 90,000 +/- 5000. BPO 1a and 1b were dimers with subunit Mr values of 34,000 and 43,000, respectively. BPO 3 was a trimer with a subunit Mr of 31,000. The enzymes differed in their isoelectric points, heat stability, and Km values. In immunodiffusion experiments BPO 1a and 3 showed partial identity with the nonhaem-type bromoperoxidase from Streptomyces aureofaciens. The nonhaem-type BPO 1a, 1b and 3 had neither peroxidase nor catalase activity.     

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.     

Haloperoxidase activities in Arctic macroalgae

Haloperoxidase activities were assayed in 21 species of Arctic green, red and brown macroalgae collected in the Kongsfjord at Ny-Ålesund (Svalbard). The enzymes were specific for bromide and/or iodide, but not for chloride. Highest bromoperoxidase activities were found in the brown seaweads Laminaria saccharina and L. digitata, whereas highest iodoperoxidase activities were measured in the green species Acrosiphonia sonderi and Enteromorpha compressa. Optimum pH for bromination ranged between pH 4 and 7.     

龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

STUDIES OF VANDADIUM-BROMOPEROXIDASE USING SURFACE AND CORTICAL PROTOPLASTS OF MACROCYSTIS PYRIEFERA (PHAEOPHYTA)1

Aqueous Tri-SO₄ buffer (pH 8.3) extracts of cortical and surface protoplasts of Macrocystis pyrifera (L.) C. Ag. Catalyzed the bromination of monochlorodimedone (2-chloro-5, 5-dimethyl-1, 3-dimedone, MCD) in the presence of hydrogen peroxide and bromide. The apparent bromo-peroxidase activity as measured by the bromination of MCD was inhibited by the presence of endogenous compounds which are probably polyphenolics compounds (i.e. polymers of phloroglucinol) or other inhibitors. The bromoperoxidase activity of the protoplast extracts increased substantially when the extracts were washed extensively with Tris-SO₄ buffer (pH 8.3) by ultrafiltration. The bromoperoxidase activity of both surface and cortical protoplast extracts was dependent on the presence of vanadium, indicating that the bromoperoxidase present in cortical and surface cells of M. pyrifera is vanadium-bromoperoxidase. Halogenated compounds constitute one of the most significant classes of marine natural products. Since bromoperoxidases are assumed to be involved in the biosynthesis of these compounds, elucidation of the location of BrPO with in the algal tissue is important.     

Purification of bromoperoxidase from Corallina pilulifera

Bromoperoxidase was purified from the crude extract of Corallina pilulifera (Corallinaeae, Rhodophyta) and found to be homogeneous upon disc gel electrophoresis by precipitation of ammonium sulfate and sequential column chromatographies of DEAE-Sepharose CL-6B, Sepharose 6B and Cellulofine GC-700m. The purified enzyme did not exhibit optical absorption spectra of a hemoprotein. Therefore, bromoperoxidase of C. pilulifera was completely distinguishable from other haloperoxidases which have heme-irons at the catalytic sites.     

Sulfoxidation mechanism of vanadium bromoperoxidase from Ascophyllum nodosum. Evidence for direct oxygen transfer catalysis.

We have previously shown that vanadium bromoperoxidase from Ascophyllum nodosum mediates production of the (R)-enantiomer of methyl phenyl sulfoxide with 91% enantiomeric excess. Investigation of the intrinsic selectivity of vanadium bromoperoxidase reveals that the enzyme catalyzes the sulfoxidation of methyl phenyl sulfide in a purely enantioselective manner. The K(m) of the enzyme for methyl phenyl sulfide was determined to be approximately 3.5 mM in the presence of 25% methanol or tert-butanol. The selectivity of the sulfoxidation of methyl phenyl sulfide is optimal in the temperature range 25-30 degrees C and can be further optimized by increasing the enzyme concentration, yielding selectivities with up to 96% enantiomeric excess. Furthermore, we established for the first time that vanadium bromoperoxidase is functional at temperatures up to 70 degrees C. A detailed investigation of the sulfoxidation activity of this enzyme using (18)O-labeled hydrogen peroxide shows that vanadium bromoperoxidase mediates the direct transfer of the peroxide oxygen to the sulfide. A schematic model of the vanadium haloperoxidase sulfoxidation mechanism is presented.     

龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。