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.     

Copper stress proteomics highlights local adaptation of two strains of the model brown alga Ectocarpus siliculosus

Ectocarpus siliculosus is a cosmopolitan brown alga with capacity to thrive in copper enriched environments. Analysis of copper toxicity was conducted in two strains of E. siliculosus isolated from (i) an uncontaminated coast in southern Peru (Es32) and (ii) a copper polluted rocky beach in northern Chile (Es524). Es32 was more sensitive than Es524, with toxicity detected at 50 μg/L Cu, whereas Es524 displayed negative effects only when exposed to 250 μg/L Cu. Differential soluble proteome profiling for each strain exposed to sub‐lethal copper levels allowed to identify the induction of proteins related to processes such as energy production, glutathione metabolism as well as accumulation of HSPs. In addition, the inter‐strain comparison of stress‐related proteomes led to identify features related to copper tolerance in Es524, such as striking expression of a PSII Mn‐stabilizing protein and a Fucoxanthine chlorophyll a–c binding protein. Es524 also expressed specific stress‐related enzymes such as RNA helicases from the DEAD box families and a vanadium‐dependent bromoperoxidase. These observations were supported by RT‐qPCR for some of the identified genes and an enzyme activity assay for vanadium‐dependent bromoperoxidase. Therefore, the occurrence of two different phenotypes within two distinct E. siliculosus strains studied at the physiological and proteomic levels strongly suggest that persistent copper stress may represent a selective force leading to the development of strains genetically adapted to copper contaminated sites.     

Vanadium-dependent bromoperoxidases from <Emphasis Type="Italic">Gracilaria</Emphasis> algae

Red algae from the Gulf of Thailand were examined for haloperoxidatic activity. Six species, Gracilaria changii, G. edulis, G. firma, G. fisheri, G. salicornia, and G. tenuistipitata, showed bromoperoxidatic activity. Duplicate polyacrylamide electrophoretic gels showed enzyme activity patterns developed by phenol red staining for bromoperoxidatic activity and by 3,3′-diaminobenzidine staining for peroxidatic activity. All algae gave isoenzymic bromoperoxidatic activity bands and peroxidatic activity bands, but there were peroxidatic and bromoperoxidatic activity bands that did not correspond. The bromoperoxidatic activity of the crude enzyme extracts as well as previously dialyzed enzyme solutions was enhanced significantly by incubation with vanadium pentoxide. The three purified bromoperoxidases from G. fisheri contained vanadium, and their relative activities corresponded to the ratio of vanadium to enzyme. In addition, they were not inhibited by H₂O₂. These data confirm that the enzymes are vanadium bromoperoxidases.     

Enhancing effect of calcium and vanadium ions on thermal stability of bromoperoxidase from Corallina pilulifera

Bromoperoxidase from the macro-alga Corallina pilulifera is an enzyme that possesses vanadate in the catalytic center, and shows a significant thermostability and stability toward organic solvents. The structural analysis of the recombinant enzyme overexpressed in yeast revealed that it contains one calcium atom per subunit. This has been confirmed by inductively coupled plasma emission spectrometry experiments. The study of the effect of metal ions on the apo-enzyme stability has shown that the calcium ion significantly increased the enzyme stability. In addition, vanadate also increased the thermostability and strontium and magnesium ions had similar effects as calcium. The holo-enzyme shows high stability in a range of organic solvents. The effect of the different ions and solvents on the structure of the enzyme has been studied by circular dichroism experiments. The high stability of the enzyme in the presence of organic solvents is useful for its application as a biocatalyst.     

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.     

超滤膜分离纯化珊瑚藻溴过氧化物酶

利用超滤膜对珊瑚藻中溴过氧化物酶(EC 1.11.1.18,分子质量740kDa)进行分离纯化,对膜的截留分子质量、操作压力、起始蛋白质浓度、搅拌速率、pH、离子强度等条件进行了优化。超滤分离纯化最优条件为:截留分子质量为100kDa的聚偏氟乙烯(PVDF)膜,操作压力为0.02MPa,搅拌速率为600r/min,起始蛋白质浓度为0.1g/L,pH=6.0。对粗酶液先进行热沉淀纯化,再进行超滤纯化,溴过氧化物酶被纯化了21倍,比活为212U/mg,酶活回收率为96%。     

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^–.     

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.