Expression of the vanadium-dependent bromoperoxidase gene from a marine macro-alga Corallina pilulifera in Saccharomyces cerevisiae and characterization of the recombinant enzyme

The vanadium-dependent bromoperoxidase from the marine macro-alga Corallina pilulifera was heterologously expressed in Saccharomyces cerevisiae. The enzyme was purified and crystals in "tear drop" form were obtained. The catalytic properties of the recombinant enzyme were studied and compared with those of the native enzyme purified from C. pilulifera. Differences in thermal stability and chloroperoxidase activity were observed. The recombinant enzyme retained full activity after preincubation at 65 degrees C for 20 min, but the native enzyme was completely inactivated under the same conditions. The chlorinating activity of the native enzyme was more than ten times higher than that of the recombinant enzyme. Other properties, such as K(m) values for KBr and H(2)O(2), and optimal temperature and pH, were similar for each source of C. pilulifera bromoperoxidase.     

Biotransformation of alkenes by haloperoxidases: Regiospecific bromohydrin formation from cinnamyl substrates

Summary A study of bromohydrin formation from some cinnamyl substrates by the heme-dependent chloroperoxidase from Caldariomyces fumago has identified differences in the regio- and diastereospecificity of this enzyme relative to chemical hydrobromination. Diastereospecificity of bromohydrin formation from (E)-4-phenyl-buten-2-ol by the vanadium-dependent bromoperoxidase from the alga Corallina officinalis is similar to that of the fungal enzyme but significantly different to the equivalent chemical reaction.     

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.     

Characterization of nonheme type bromoperoxidase in Corallina pilulifera

Bromoperoxidase was purified from the crude extract of Corallina pilulifera to be homogeneous upon polyacrylamide disc gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses according to the procedures previously reported (Itoh, N., Izumi, Y., and Yamada, H. (1985) Biochem. Biophys. Res. Commun. 131, 428-435). The enzyme had a molecular weight of approximately 790,000 and was composed of 12 subunits of identical molecular weights (Mr 64,000). Hexagonal molecular shapes of the enzyme were observed by electron microscopy. The isoelectric point of the enzyme was 3.0, and the predominance of acidic amino acids was revealed by amino acid analysis of the enzyme. The enzyme was specific for I- and Br- and inactive toward Cl- and F-. The optimum pH of the enzyme was 6.0, and the enzyme was stable in a range from pH 5.0 to 11.0. The enzyme had no hemeor flavin-like compounds as a prosthetic group. Plasma emission spectroscopy revealed that the enzyme contains 2.3 +/- 0.2 atoms of iron and 1.6 +/- 0.1 atoms of magnesium/molecule of protein. Hence, bromoperoxidase of C. pilulifera was distinct from other haloperoxidases and many peroxidases, which are hemoproteins.     

Vanadate activation of bromoperoxidase from Corallina officinalis

A nonheme bromoperoxidase has been purified to homogeneity from the red seaweed Corallina officinalis. Like the corresponding enzyme previously reported from C. pilulifera, this bromoperoxidase contains a significant amount of nonheme iron. However, it is vanadate ion and not iron that activates the enzyme, and maximal activity is achieved with stoichiometric vanadium incorporation. The absence of competition between vanadium and iron suggests that they occupy distinct binding sites in the protein. A correlation between vanadium content and catalytic activity indicates that less than 12 percent of the maximal activity of the enzyme can be derived from metals other than vanadium.     

Enzymatic bromination of barbituric acid and some of its derivatives

Barbituric acid, 1-methyl- and 1,3-dimethylbarbituric acid, some of its 5-phenyl derivatives, and 5-chlorobarbituric acid are presented as new substrates for the bromoperoxidase isolated from the brown alga Ascophyllum nodosum. This enzyme is able to convert these substrates into the corresponding 5-bromo or 5,5-dibromo derivatives in good yields. Kinetic measurements show that the structure of the examined substrates has little or no effect on the enzymatic rate of bromination. However, at low substrate concentration the reaction rate depends on both the concentration of the organic substrate and the concentration of hydrogen peroxide. A mechanism is proposed for the reactions of bromoperoxidase with its substrates. These reactions involve the formation of free hypobromous acid which can either brominate the organic halogen acceptor or produce singlet oxygen by a competing reaction with hydrogen peroxide.     

Cloning and biochemical characterization of Co(2+)-activated bromoperoxidase-esterase (perhydrolase) from Pseudomonas putida IF-3 strain

The gene encoding Co(2+)-activated bromoperoxidase (BPO)-esterase (EST), catalyzing the organic acid-assisted bromination of some organic compounds with H2O2 and Br(-) and quite specific hydrolysis of (R)-acetylthioisobutyric acid methyl ester, was cloned from the chromosomal DNA of the Pseudomonas putida IF-3 strain. The bpo-est gene comprises 831 bp and encoded a protein of 30181 Da. The enzyme was expressed at a high level in Escherichia coli and purified to homogeneity by ammonium sulfate fractionation and two-step column chromatographies. The recombinant enzyme required acetic acid, propionic acid, isobutyric acid or n-butyric acid in addition to H2O2 and Br(-) for the brominating reaction and was activated by Co(2+) ions. It catalyzed the bromination of styrene and indene to give the corresponding racemic bromohydrin. Although the enzyme did not release free peracetic acid in the reaction mixture, chemical reaction with peracetic acid could well explain such enzymatic reactions via a peracetic acid intermediate. The results indicated that the enzyme was a novel Co(2+)-activated organic acid-dependent BPO (perhydrolase)-EST, belonging to the non-metal haloperoxidase-hydrolase family.     

Bromoperoxidase activity in microplantlet suspension cultures of the macrophytic red alga Ochtodes secundiramea.

Bromoperoxidase is an enzyme found in marine macroalgae that catalyzes the bromination of organic substrates. Photosynthetic microplantlet suspension cultures derived from the macrophytic red alga Ochtodes secundiramea were shown to possess bromoperoxidase. The optimum pH for O. secundiramea bromoperoxidase activity in cell-free extracts was 6.0, and the half-saturation constant for bromination of the exogeneous substrate monochlorodimedone (MCD) was 18 microM. O. secundiramea microplantlets were cultivated in a bubble-column photobioreactor at an incident light intensity of 38 microE x m(-2) x s(-1) (71% of light-saturated photosynthesis, 10:14 light:dark photoperiod), and the kinetics of cell growth and bromoperoxidase production were followed. At these conditions, the specific growth rate was 0.052 x day(-1). The lowest specific bromoperoxidase activity of 0.3 micromol MCD x g(-1) cell x min(-1) occurred during the midexponential phase of growth, and then increased steeply to 1.9 micromol MCD x g(-1) cell x min(-1) during the late stationary phase, suggesting that bromoperoxidase production was part of secondary metabolism. The estimated bromoperoxidase content in the cell mass at late stationary phase was 67 microg x g(-1) dry cell mass, demonstrating that bioreactor production of marine bromoperoxidase is feasible.     

Modification of halogen specificity of a vanadium-dependent bromoperoxidase

The halide specificity of vanadium-dependent bromoperoxidase (BPO) from the marine algae, Corallina pilulifera, has been changed by a single amino acid substitution. The residue R397 has been substituted by the other 19 amino acids. The mutant enzymes R397W and R397F showed significant chloroperoxidase (CPO) activity as well as BPO activity. These mutant enzymes were purified and their properties were investigated. The maximal velocities of CPO activities of the R397W and R397F enzymes were 31.2 and 39.2 units/mg, and the K(m) values for Cl(-) were 780 mM and 670 mM, respectively. Unlike the native enzyme, both mutant enzymes were inhibited by NaN(3). In the case of the R397W enzyme, the incorporation rate of vanadate into the active site was low, compared with the R397F and the wild-type enzyme. These results supported the existence of a specific halogen binding site within the catalytic cleft of vanadium haloperoxidases.     

Characterization of nonheme iron and reaction mechanism of bromoperoxidase in Corallina pilulifera

The properties of the nonheme iron of bromoperoxidase from Corallina pilulifera were studied. The enzyme lost its activity when reduced with formamidine-sulfinic acid and recovered it when oxidized by air. Incubation of the enzyme with ferric or ferrous ion-chelating agents indicated that its nonheme iron was ferric. Analyses of circular dichroism and proton NMR spectra suggested that the ferric ion tightly bound to cysteine, histidine, or tyrosine residues of the enzyme. The enzyme catalyzed Br--dependent catalase reactions to yield 1 mol of O2 from 2 mol of H2O2. No O2 evolution was observed when bromination reaction of monochlorodimedone occurred. From these results, together with previous knowledge of this enzyme, it was concluded that it activated bromide anion (Br-) to bromonium cation (Br+) using one molecule of H2O2, and this Br+OH- formed at the active site then decomposed another H2O2 to yield O2 in the absence of halogen acceptors (substrate). When substrate was present in the reaction mixture, it and H2O2 competitively reacted with the reaction intermediate (Br+OH-) to give brominated products.     

Interaction of hypohalous acids and heme peroxidases with unsaturated phosphatidylcholines

The formation of chlorohydrins, bromohydrins, and iodohydrins from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by the myeloperoxidase-hydrogen peroxide-halide system was evaluated by means of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. This approach allows to detect different kinds of the halogenation reaction even in one mass spectrum. Using a mixture of Cl-, Br-, I-, and SCN- at physiological concentrations, a bromination of POPC dominates by the MPO-hydrogen peroxide-halide system. Hypothiocyanite does apparently not react with the double bond of POPC, but increasing amounts of SCN- cause a decrease of the bromohydrin peaks. An interconversion between different hypohalous acids produced by the myeloperoxidase-hydrogen peroxide-halide system determines the pattern of halogenohydrins in POPC. Especially, hypochlorous acid is able to oxidise Br- to hypobromous acid.     

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.     

Microbial production of antibiotic fosfomycin by a stereoselective epoxidation and its formation mechanism

Fifteen strains of aerobic bacteria and two actinomycetous strains were isolated from various soil samples, using cis-propenylphosphonic acid as the sole source of carbon. Taxonomic studies showed that these belonged to the Pseudomonas, Alcaligenes, Flavobacterium, Aeromonas, Corynebacterium and Streptomyces genera. Most of these strains produced the antibiotic fosfomycin [(-)-(1R, 2S)-1,2-epoxypropylphosphonic acid] from cis-propenylphosphonic acid by stereoselective epoxidation. Under the culture conditions tested, the production of fosfomycin reached a level of 2.0 mg/ml and 0.75 mg/ml by F. esteroaromaticum IFO 3751 and P. putida IK-8 respectively. Purified fosfomycin was an active stereoisomer and its optical purity was a little higher than that of the synthetic compound. Although the stereoespecificity of each enzyme participating in the epoxidation reaction has not yet been clarified, the epoxide formation proceeded through a bromohydrin intermediate produced by bromoperoxidase, finally yielding fosfomycin by the action of halohydrin epoxidase.     

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。     

The chlorination of barbituric acid and some of its derivatives by chloroperoxidase

Barbituric acid and some of its derivatives are presented as new substrates for the chloroperoxidase from Caldariomyces fumago. These compounds are rapidly converted to the 5-chloro or 5,5-dichloro derivatives, in very high yield. The reaction path is discussed and the kinetics of the reactions are investigated. It is shown that neither the concentration nor the structure of the organic substrate has any influence on the rate of halogenation. The enzymatic chlorination of 1-methyl-5-phenylbarbituric acid does not proceed in a stereoselective manner. The results are compared with the present theories concerning the enzymatic reaction mechanism, and the current research on this topic is evaluated. The available data do not as yet permit a definitive choice of reaction mechanism.     

Addition reactions at the 16(17) double bond of 3-methoxy-13alpha-estra-1,3,5(10),16-tetraene

The epoxidation, the addition of hypobromous acid, and the hydroboration of 3-methoxy-13alpha-estra-1,3,5(10),16-tetraene 1 with diborane, catecholborane, and 9-BBN were investigated in order to determine the stereochemical outcome and to synthesize new 13alpha-estra-1,3,5(10)-trienes for biological and conformational investigations. It was shown that the sterically demanding reagent 9-BBN participated in a preferred beta attack (53% 16betaOH 10, 34% 17betaOH 8, 13% 16alphaOH 11). This stereochemical result is in agreement with that from another cis addition reaction, the recently described OsO4 dihydroxylation of 1 [Steroids 68 (2003) 113]. With smaller reagents such as B2H6, catecholborane, or magnesium monoperoxyphthalate, a diminished stereoselectivity was observed with only a slight excess of beta attack. The ionic trans addition of hypobromous acid gave two 17-bromo-16-alcohols with 16beta,17alpha (4, 76%) and 16alpha,17beta configuration (5, 24%) formed by trans cleavage of the 16,17alpha- and beta-bromonium ion at position 16. The same regioselective and stereoselective course was found for the cleavage of the 16alpha,17alpha- and 16beta,17beta-epoxides (3 and 2) with hydrazoic acid (3-->16betaN3,17alphaOH 7, 2-->16alphaN3,17betaOH 6). The stereochemistry of the addition reactions to 1 can be explained in terms of a twist-boat conformation involving the C ring of compound 1. From a synthetic viewpoint the synthesis of the beta-epoxide 2 from the bromohydrin 4, the cleavage of this epoxide to 16alpha-substituted-17beta-hydroxy compounds, such as 6, and hydroboration/oxidation with 9-BBN to the hitherto unknown 16beta-hydroxy compound 10 are useful procedures. The bromohydrin 5 is the first 13alpha-steroid with a 17beta-bromo substituent. X-ray analysis revealed twist-boat and 16beta-envelope conformations for rings C and D, respectively.     

Halogenation and Oxidation Reactions with Haloperoxidases

Haloperoxidases are enzymes which catalyze the incorporation of halogen atoms into organic molecules. They are found throughout nature, playing a major role in the defence system of many organisms. Their reaction mechanisms as well as their use as catalysts for halogenation and oxidation reactions on laboratory and industrial scales are discussed. Up to now, selective halogenation reactions have only been reported for the chloroperoxidase from Pseudomonas pyrrocinia. The usefulness of the other enzymes is based on their ability to produce hypohalous acid (HOX) in a controllable way, allowing the smooth (yet nonselective) halogenation of electron-rich substrates. On the other hand, it has been shown recently that some haloperoxidases can stereoselectively convert sulfides and alkenes into their corresponding homochiral oxides. Therefore, these enzymes will undoubtedly gain importance in the near future.     

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。     

A monoclonal antibody recognizing the chlorohydrin derivatives of oleic acid for probing hypochlorous acid involvement in tissue injury

Summary

A monoclonal antibody against hypochlorous acid—modified oleic acid has been raised to investigate involvement of HOCI in tissue injury. Mice were immunized with an isomeric mixture of chlorohydrin derivatives of oleic acid (18:0-chlorohydrin) conjugated to keyhole limpet haemocyanin (CH-KLH). The chlorohydrin was formed by the treatment of oleic acid with hypochlorous acid. Monoclonal antibodies were raised and the fusion was screened with 18:0-chlorohydrin-bovine serum albumin (CH-BSA) conjugate. A number of antibody-secreting clones were identified and the supernatants were characterized by binding studies and dose-response curves. In ELISA, mAb CH-1 had an equivalent titre when either the chlorohydrin or bromohydrin derivative of oleic acid, complexed to bovine serum albumin, was used as screening antigen. The mAb CH-1 recognition of CH-BSA was competed with chlorohydrin and bromohydrin conjugates of BSA and KLH. Similarly, free 18:0-chlorohydrin and the 18:0-chlorohydrin-phosphatidyl choline treated with hypochlorous acid competed with mAb CH-1 binding. The mAb CH-1 also recognised the chlorohydrin derivative of linoleic acid and chlorohydrin formed from palmitoyl, oleyl phosphatidyl choline but with a decreased avidity. Weak cross-reactivity was observed with hydroxy-linoleic acid and linoleic acid hdroperoxide, either as free fatty acid or in phosphatidyl choline. There was minimal competitive binding of mAb CH-1 to free oleic acid, 16:0/18:1 phosphatidylcholine, cholesterol, or cholesterol chlorohydrin.

The mAb CH-1 described here may be a useful probe for assessing the involvement of hypochlorous acid in tissue injury.     

The novel non-heme vanadium bromoperoxidase from marine algae: Phosphate inactivation

Summary Vanadium bromoperoxidase is a naturally occurring vanadium-containing enzyme isolated from marine algae. V-BrPO catalyzes the oxidation of halides by hydrogen peroxide which can result in the halogenation of organic substrates. Bromoperoxidase activity is measured by the halogenation of monochlorodimedone (2-chloro-5,5-dimethyl-1,3-dimedone, MCD). In the absence of an organic substrate, V-BrPO catalyzes the halide-assisted disproportionation of hydrogen peroxide yielding dioxygen. The dioxygen formed is in the singlet excited state (¹O₂). V-BrPO is quite stable to thermal denaturation and denaturation by certain organic solvents which makes V-BrPO an excellent candidate for industrial applications. The stability of V-BrPO in the presence of strong oxidants and in the presence of phosphate is reported. Incubation of V-BrPO in phosphate buffer (1–100 mM at pH 6; 2–10 mM at pH 5) inactivates the enzyme. The inactivity can be fully restored by the addition of vanadate if excess phosphate is removed. The inactivation of V-BrPO by phosphate can be prevented by the presence of H₂O₂ (4–40 mM). We are currently investigating the mechanism of V-BrPO inactivation by phosphate. V-BrPO was not inactivated by HOCl (1 mM) nor H₂O₂. In addition V-BrPO was not inactivated under turnover conditions of 1 mM H₂O₂ with 0.1–1 M Cl^– at pH 5 nor 2 mM H₂O₂ with 0.1 M Br^–.