Vanadium Haloperoxidases Research Articles

Insights into the Mechanism of a Vanadium Bromoperoxidase from the Marine Macro‐Algae Corallina pilulifera for Biocatalytic Halogenation

AbstractVanadium haloperoxidases have been studied to understand their mechanism and halide specificity. Crystal structures of vanadium bromoperoxidase enzyme complexes from Corallina piluifera, with vanadate and bromide and with phosphate and chloride, show significant displacement of loop residues 336–338 upon halogen binding. This shows a “closed position” of Leu337 locking the bromide ion in a hydrophobic environment favoring the vanadium peroxide reaction with the halogen by retaining the resulting hypobromite in the enzyme active site. The bound cofactor exists as a mixture of free vanadate and histidine553‐vanadate adduct. A mutant enzyme Arg397Trp also has been crystallized with bound phosphate and its structure determined with and without the bound bromide ion. The precise positions of the bromine have been determined using its anomalous signal. The bromide binding site in the mutant enzyme is displaced by 2.5 Å resulting in a mixed population of the “open” and the “closed” forms of Leu337. This allows additional chloroperoxidase activity due to re‐positioning of the halogen ion 0.6 Å closer to the vanadate ion. These studies support the application of vanadium haloperoxidase enzymes for selective halogenation of important drug molecules.

MAR4 Streptomyces: A Unique Resource for Natural Product Discovery.

Marine-derived Streptomyces have long been recognized as a source of novel, pharmaceutically relevant natural products. Among these bacteria, the MAR4 clade within the genus Streptomyces has been identified as metabolically rich, yielding over 93 different compounds to date. MAR4 strains are particularly noteworthy for the production of halogenated hybrid isoprenoid natural products, a relatively rare class of bacterial metabolites that possess a wide range of biological activities. MAR4 genomes are enriched in vanadium haloperoxidase and prenyltransferase genes, thus accounting for the production of these compounds. Functional characterization of the enzymes encoded in MAR4 genomes has advanced our understanding of halogenated, hybrid isoprenoid biosynthesis. Despite the exceptional biosynthetic capabilities of MAR4 bacteria, the large body of research they have stimulated has yet to be compiled. Here we review 35 years of natural product research on MAR4 strains and update the molecular diversity of this unique group of bacteria.

Vanadium haloperoxidases as noncanonical terpene synthases.

Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize vanadate, an aqueous halide ion, and hydrogen peroxide to produce an electrophilic halogen species that can be incorporated into electron rich organic substrates. This halogen species can react with terpene substrates and trigger halonium-induced cyclization in a manner reminiscent of class II terpene synthases. While not all VHPOs act in this capacity, several notable examples from algal and actinobacterial species have been characterized to catalyze regio- and enantioselective reactions on terpene and meroterpenoid substrates, resulting in complex halogenated cyclic terpenes through the action of single enzyme. In this article, we describe the expression, purification, and chemical assays of NapH4, a difficult to express characterized VHPO that catalyzes the chloronium-induced cyclization of its meroterpenoid substrate.

How Is Substrate Halogenation Triggered by the Vanadium Haloperoxidase from Curvularia inaequalis?

Vanadium haloperoxidases (VHPOs) are unique enzymes in biology that catalyze a challenging halogen transfer reaction and convert a strong aromatic C-H bond into C-X (X = Cl, Br, I) with the use of a vanadium cofactor and H2O2. The VHPO catalytic cycle starts with the conversion of hydrogen peroxide and halide (X = Cl, Br, I) into hypohalide on the vanadate cofactor, and the hypohalide subsequently reacts with a substrate. However, it is unclear whether the hypohalide is released from the enzyme or otherwise trapped within the enzyme structure for the halogenation of organic substrates. A substrate-binding pocket has never been identified for the VHPO enzyme, which questions the role of the protein in the overall reaction mechanism. Probing its role in the halogenation of small molecules will enable further engineering of the enzyme and expand its substrate scope and selectivity further for use in biotechnological applications as an environmentally benign alternative to current organic chemistry synthesis. Using a combined experimental and computational approach, we elucidate the role of the vanadium haloperoxidase protein in substrate halogenation. Activity studies show that binding of the substrate to the enzyme is essential for the reaction of the hypohalide with substrate. Stopped-flow measurements demonstrate that the rate-determining step is not dependent on substrate binding but partially on hypohalide formation. Using a combination of molecular mechanics (MM) and molecular dynamics (MD) simulations, the substrate binding area in the protein is identified and even though the selected substrates (methylphenylindole and 2-phenylindole) have limited hydrogen-bonding abilities, they are found to bind relatively strongly and remain stable in a binding tunnel. A subsequent analysis of the MD snapshots characterizes two small tunnels leading from the vanadate active site to the surface that could fit small molecules such as hypohalide, halide, and hydrogen peroxide. Density functional theory studies using electric field effects show that a polarized environment in a specific direction can substantially lower barriers for halogen transfer. A further analysis of the protein structure indeed shows a large dipole orientation in the substrate-binding pocket that could enable halogen transfer through an applied local electric field. These findings highlight the importance of the enzyme in catalyzing substrate halogenation by providing an optimal environment to lower the energy barrier for this challenging aromatic halide insertion reaction.

Decarboxylative Bromooxidation of Indoles by a Vanadium Haloperoxidase

Halooxindoles are versatile building blocks for the construction of complex oxindole-containing targets of biological importance. Despite their synthetic value, catalytic methods to make 3-halooxindoles from readily available starting materials have remained undisclosed. We recently discovered that the chloroperoxidase from Curvularia inaequalis (CiVCPO) is a viable catalyst for the decarboxylative bromooxidation of 3-carboxyindoles to furnish 3-bromooxindoles with excellent regio- and chemoselectivity. In addition to the development of the synthetic method, this study provides evidence of a bromide recycling mechanism for the indole oxidation event. A discussion of the reaction development, substrate scope, mechanistic insights, and reaction applicability will be discussed herein.

Synthesis of oxidovanadium complexes containing coumarin and naphthalene: bromoperoxidase activity and DNA/BSA binding

Two tridentate Schiff base ligands, L1 and L2 , have been introduced in the synthesis of mononuclear oxidovanadium complexes with a co-ligand 1,10-phenanthroline. Two mononuclear complexes, [VO(L1)(1,10-phen)] (1) and [VO(L2)(1,10-phen)] (2), have been synthesized in high yields by reactions with [VO(acac)2] in 1:1 ratio in methanol under reflux with 1,10-phenanthroline as co-ligand. X-ray crystallographic studies unveil the structures of 1 and 2 showing L1 and L2 bind in O^N^O coordination. The complexes were characterized by using UV-Vis, IR, NMR and Mass spectral techniques. The physiochemical properties have been interpreted by density functional theory (DFT) and time dependent density functional theory (TDDFT) calculations. The mimicking of vanadium haloperoxidase was investigated by bromination of phenol red in the presence of bromide and H2O2 with a rate of 2.68 × 102 (mol/L)−2 s−1. The DNA and BSA protein binding interactions of 1 and 2 have been explored by UV–Vis and fluorescence spectral methods; viscosity measurements reveal that the complexes interact with CT-DNA through intercalation in the order [VO(L1)(1,10-phen)](2.71 × 104) > [VO(L2)(1,10-phen)](9.67 ×103). The complexes exhibit binding interactions with BSA protein.

Decarboxylative halogenation of indoles by vanadium haloperoxidases.

Halogenated heteroarenes are key building blocks across numerous chemical industries. Here, we report that vanadium haloperoxidases are capable of producing 3-haloindoles through decarboxylative halogenation of 3-carboxyindoles. This biocatalytic method is applicable to decarboxylative chlorination, bromination, and iodination in moderate to high yields and with excellent chemoselectivity.

Single-atom tungsten engineering of MOFs with biomimetic antibiofilm activity toward robust uranium extraction from seawater

Selective uranium extraction from seawater is challenging because of ubiquitous marine biofouling and poor reusability of adsorbents. A reusable atomic-scale tungsten nanozyme integrating uranium capture capacity and intrinsic haloperoxidase-like activity is constructed, resulting in strong antibiofouling ability and superb reusability for uranium extraction after consecutive multiple regeneration cycles in bio-aggressive seawater. • Atomically dispersed tungsten is anchored in metal organic frameworks (W-UiO). • The presence of atomic-scale tungsten endows W-UiO with haloperoxidase-mimicking activity. • W-UiO nanozyme exerts strong antibacterial activity and inhibition effect against marine microbial colonization. • Such nanozyme exhibits excellent reusability for uranium extraction from seawater. Uranium is the essential element for nuclear power industry. Selective uranium extraction from seawater is very attractive, but challenging because ubiquitous marine biofouling seriously impairs the uranium extraction capacity and reusability of absorbents in bio-aggressive seawater. Herein, we construct a reusable nanozyme for uranium recovery from seawater based on atomically dispersed tungsten anchored on metal organic frameworks (W-UiO). We illustrate that W-UiO nanozyme mimics naturally occurring vanadium haloperoxidase that catalytically converting halides to biocidal hypobromous acid, inducing strong antibiofouling activity against marine microbial colonization. As demonstrated, W-UiO nanozyme shows superb reusability in raw seawater simultaneously, preserving a 78.0% of its initial uranium capture capacity (6.8 times higher than its pristine counterpart) after five consecutive adsorption-desorption cycles. This study is anticipated to spur rationally realizing robust absorbents by mimicking the natural enzymes for practical application in uranium recovery from seawater.

Evidence for an N-Halohistidyl Intermediate in the Catalytic Cycle of Vanadium Chloroperoxidase (VCPO) and an Artificial Enzyme Derived from VCPO: A Computational Investigation

Vanadium haloperoxidases play an important catalytic role in the natural production of antibiotics which are difficult to make in the laboratory. Understanding the catalytic mechanism of these enzymes will aid in the production of artificial enzymes useful in bioengineering the synthesis of drugs and useful chemicals. However, the catalytic mechanism remains not fully understood yet. In this paper, we investigate one of the key steps of the catalytic mechanism using QM/MM. Our investigation reveals a new N-halohistidyl intermediate in the catalytic cycle of vanadium chloroperoxidase (VCPO). This new intermediate, in turn, can explain the known inhibition of the enzyme by substrate under acidic conditions (pH[Formula: see text]4). Additionally, we examine the possibility of replacing V in VCPO by Nb or Ta using QM modeling. We report the new result that the Gibbs free energy barriers of several steps of the catalytic cycle are lower in the case of artificial enzymes, incorporating NbO[Formula: see text] or TaO[Formula: see text] instead of VO[Formula: see text]. Our results suggest that these new artificial enzymes may catalyze the oxidation of halide faster than the natural enzyme.

How Do Vanadium Chloroperoxidases Generate Hypochlorite from Hydrogen Peroxide and Chloride? A Computational Study

Vanadium haloperoxidases are one of the few enzymes in nature that utilize a vanadium center and catalyze the halogenation of substrates through the biosynthesis of hypohalite. Vanadium chloroperox...

Tungstate-Catalyzed Biomimetic Oxidative Halogenation of (Hetero)Arene under Mild Condition.

SummaryAryl halide (Br, Cl, I) is among the most important compounds in pharmaceutical industry, material science, and agrochemistry, broadly utilized in diverse transformations. Tremendous approaches have been established to prepare this scaffold; however, many of them suffer from atom economy, harsh condition, inability to be scaled up, or cost-unfriendly reagents and catalysts. Inspired by vanadium haloperoxidases herein we presented a biomimetic approach for halogenation (Br, Cl, I) of (hetero)arene catalyzed by tungstate under mild pH in a cost-efficient and environment- and operation-friendly manner. Broad substrates, diverse functional group tolerance, and good chemo- and regioselectivities were observed, even in late-stage halogenation of complex molecules. Moreover, this approach can be scaled up to over 100 g without time-consuming and costly column purification. Several drugs and key precursors for drugs bearing aryl halides (Br, Cl, I) have been conveniently prepared based on our approach.

Role of Local Structure on Catalytic Reactivity: Comparison of Methanol Oxidation by Aqueous Bioinorganic Enzyme Mimic (Vanadium Haloperoxidase) and Vanadia-Based Heterogeneous Catalyst (Supported VO4/SiO2)

Although enzymes perform redox reactions at milder reaction conditions than heterogeneous solid catalysts, the origin of this reactivity difference still needs to be resolved. In the present study,...

Second-Coordination Sphere Effect on the Reactivity of Vanadium-Peroxo Complexes: A Computational Study.

Vanadium-oxo and vanadium-peroxo complexes are common intermediates in biology and are, for instance, found in the catalytic cycle of vanadium haloperoxidases. In biomimetic chemistry synthetic models have been created that mimic the structural features of the coordination environment of these vanadium-oxo and vanadium-peroxo species. Recently, two novel vanadium-oxo complexes were trapped and characterized with a trigonal bipyramidal ligand design with either a solvent exposed vanadium center or the vanadium inside a cage, designated as the bowl-shaped configuration and the dome-shaped structure, respectively. Density functional theory calculations are reported here on these bowl- and dome-shaped structures where we study the reaction with t-butylhydroperoxide to form the vanadium-peroxo species and its reaction with thioanisole. Although the structural features of the vanadate core are close for both structures, the calculations display a strong second-coordination sphere effect of the ligand architecture on the barrier heights of the reaction with a terminal oxidant even though the rate-determining transition states show little structural differences. A similar observation is seen for the reaction of the two vanadium-peroxo species with thioanisole. Overall, the calculations implicate that vanadium-peroxo is an efficient oxidant of sulfoxidation reactions, although it is not as efficient as analogous iron(IV)-oxo heme and nonheme oxidants that react with substantially lower barriers. The reactivity differences are analyzed with thermochemical cycles and valence bond patterns that explain the differences in chemical properties and identify how the ligands affect the chemical reactivity with substrates.

Bioinorganic Laboratory Experiment: Synthesis and Catalytic Activity of a Vanadium Haloperoxidase Model Complex

Laboratory experiments that offer interdisciplinary experiences for students are appealing and are increasingly popular additions to undergraduate chemistry curricula. Students can capitalize on their knowledge of multiple areas of chemistry while working through an application, and this fosters the development of progressive problem-solving skills. In this laboratory experiment, students combine the fields of inorganic chemistry with biological chemistry to synthesize and test the catalytic activity of a model of vanadium haloperoxidase, an enzyme naturally produced in certain brown- and red-seaweed species. Students use 1H NMR spectroscopy to monitor the oxidation of thioanisole to methyl phenyl sulfoxide using their prepared vanadium complex as the catalyst.

Two dinuclear oxidovanadium(V) complexes of N2O2 donor amine-bis(phenolate) ligands with bromo-peroxidase activities: Kinetic, catalytic and computational studies

Two dinuclear oxidovanadium(V) complexes [LiVVO(µ2-O)VVO(Li)] (i = 1, H2L1, complex 1 and i = 2 for H2L2, complex 2) of two ONNO donor amine-bis(phenolate) ligands have been synthesized and characterized by X-ray diffraction studies which exhibited distorted octahedral geometry around each V center. In MeCN the complexes exist as dimers as indicated by HRMS studies, however, in the presence of 2 or more equivalents of H+ the dimers turned into monomers, ([LiVV = O]+ which exists in equilibrium with ([LiVV = OH]2+ and evidenced from the shift in λmax from 685 nm to 765 nm for complex 1 and 600 to 765 nm for complex 2. The complexes 1 and 2 efficiently catalyze the oxidative bromination of salicylaldehyde in the presence of H2O2 to produce 5-bromo-salicylaldehyde as the major product with TONs 405 and 450, respectively in the mixed solvent system (H2O:MeOH:THF = 4:3:2, v/v). The kinetic analysis of the bromide ion oxidation reaction indicates a mechanism which is first order in peroxidovanadium complex and bromide ion and limiting first-order on [H+]. The evaluated kBr and kH values are (8.82 ± 0.35) and (65.0 ± 2.23) M−1 s−1 for complex 1 and (6.74 ± 0.19) and (61.87 ± 2.27) M−1 s−1 for complex 2, respectively. The Ka of protonated species ([LiVV = OH]2+ are: Ka = (4.3 ± 0.40) × 10−3 (pKa = 2.37) and (4.7 ± 0.50) × 10−3 (pKa = 2.33) for complex 1 and 2 respectively. On the basis of the chemistry displayed by these model compounds, a mechanism of bromide oxidation and a tentative catalytic cycle have been framed which might be relevant to vanadium haloperoxidase enzymes and supported by DFT calculations.

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 40years 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).

Emission of volatile halogenated compounds, speciation and localization of bromine and iodine in the brown algal genome model Ectocarpus siliculosus

This study explores key features of bromine and iodine metabolism in the filamentous brown alga and genomics model Ectocarpus siliculosus. Both elements are accumulated in Ectocarpus, albeit at much lower concentration factors (2-3 orders of magnitude for iodine, and < 1 order of magnitude for bromine) than e.g. in the kelp Laminaria digitata. Iodide competitively reduces the accumulation of bromide. Both iodide and bromide are accumulated in the cell wall (apoplast) of Ectocarpus, with minor amounts of bromine also detectable in the cytosol. Ectocarpus emits a range of volatile halogenated compounds, the most prominent of which by far is methyl iodide. Interestingly, biosynthesis of this compound cannot be accounted for by vanadium haloperoxidase since the latter have not been found to catalyze direct halogenation of an unactivated methyl group or hydrocarbon so a methyl halide transferase-type production mechanism is proposed.

Direct detection and characterization of bioinorganic peroxo moieties in a vanadium complex by 17O solid-state NMR and density functional theory

Electronic and structural properties of short-lived metal-peroxido complexes, which are key intermediates in many enzymatic reactions, are not fully understood. While detected in various enzymes, their catalytic properties remain elusive because of their transient nature, making them difficult to study spectroscopically. We integrated 17O solid-state NMR and density functional theory (DFT) to directly detect and characterize the peroxido ligand in a bioinorganic V(V) complex mimicking intermediates non-heme vanadium haloperoxidases. 17O chemical shift and quadrupolar tensors, measured by solid-state NMR spectroscopy, probe the electronic structure of the peroxido ligand and its interaction with the metal. DFT analysis reveals the unusually large chemical shift anisotropy arising from the metal orbitals contributing towards the magnetic shielding of the ligand. The results illustrate the power of an integrated approach for studies of oxygen centers in enzyme reaction intermediates.

Enzymatic delignification and hexenuronic acid removal in cellulosic papermaking pulp using a haloperoxidase

A novel approach for lignin and hexenuronic acid removal from cellulosic pulp based on the combination of a vanadium haloperoxidase and a tertiary amine co-catalyst.

DNA/BSA binding interactions and VHPO mimicking potential of vanadium(IV) complexes: Synthesis, structural characterization and DFT studies

Vanadium(IV) Schiff base complexes (VOL1‐VOL3) were synthesized and characterized by elemental analysis, various spectral methods and single crystal XRD studies. Structural analysis of VOL2 reveals that the central vanadium ion in the complex is six coordinate with distorted octahedral geometry. Density functional theory (DFT) and time dependent (TD‐DFT) studies were used to understand the electronic transitions observed in the complexes in UV–Vis spectra. The electrochemical behavior of the complexes was investigated in acetonitrile medium exhibit quasi‐reversible one electron transfer. The DNA and BSA protein binding interaction of vanadium complexes has been explored by UV–Vis and fluorescence spectral methods and viscosity measurements reveal that the complexes interact with CT‐DNA through intercalation mode and follows the order VOL1 &lt; VOL3 &lt; VOL2. The complexes exhibit binding interactions with BSA protein. The complexes act as chemical nuclease and cleave DNA in the presence of H2O2. The 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay was used to evaluate the radical scavenging activity demonstrate the antioxidant property of the complexes. The antimicrobial activity was screened for several microorganisms, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli. The mimicking of vanadium haloperoxidase was investigated by the bromination of the organic substrate phenol red by vanadium complexes in the presence of bromide and H2O2.