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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.

Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using,, hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change-oxidation state during the reaction. We employed an NMR. crytallography approach based on V-51 magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO), The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic-activity are examined at various pH values. Optimal sensitivity attained due to-the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate. cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated atpH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and Mutant VCPO and their pH-dependence. This study demonstrates that V-51-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.

The vanadium-dependent chloroperoxidase from Curvularia inaequalis is a stable and efficient biocatalyst for the hydroxyhalogenation of a broad range of alkenes into halohydrins. Up to 1 200 000 TON with 69 s−1 TOF were observed for the biocatalyst. A bienzymatic cascade to yield epoxides as reaction products is presented.

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144±10μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.

A catalytic, enzyme-initiated (aza-) Achmatowicz reaction is presented. The involvement of a robust vanadium-dependent peroxidase from Curvularia inaequalis allows the simple use of H2O2 and catalytic amounts of bromide.

Vanadium K -edge X-ray Absorption Spectra have been recorded for the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis at pH 6.0. The Extended X-ray Absorption Fine Structure (EXAFS) regions provide a refinement of previously reported crystallographic data; one short V = O bond (1.54 A) is present in both forms. For the native enzyme, the vanadium is coordinated to two other oxygen atoms at 1.69 A, another oxygen atom at 1.93 A and the nitrogen of an imidazole group at 2.02 A. In the peroxo-form, the vanadium is coordinated to two other oxygen atoms at 1.67 A, another oxygen atom at 1.88 A and the nitrogen of an imidazole group at 1.93 A. When combined with the available crystallographic and kinetic data, a likely interpretation of the EXAFS distances is a side-on bound peroxide involving V–O bonds of 1.67 and 1.88 A; thus, the latter oxygen would be ‘activated’ for transfer. The shorter V–N bond observed in the peroxo-form is in line with the previously reported stronger binding of the cofactor in this form of the enzyme. Reduction of the enzyme with dithionite has a clear influence on the spectrum, showing a change from vanadium(V) to vanadium(IV).

The aim of this study was to investigate the antimicrobial activity of vanadium chloroperoxidase (VCPO) reaction products on planktonic and biofilm cellsof Streptococcus mutans C180–2. Planktonic and biofilm cells were incubated in a buffered reaction mixture containing VCPO, halide (either chloride or bromide) and hydrogen peroxide, and the killing efficacy was assessed by CFU counts. The enzymatic products formed by VCPO significantly reduced the viability of planktonic and biofilm cells compared to their negative controls and the effect on the biofilm cells was more effective than a 0.2% chlorhexidine digluconate treatment. We conclude that VCPO and its reaction products form a potent antimicrobial system against S. mutans.

Aims: Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H2O2 concentration that is low compared to current nonenzymatic formulations.Methods and Results: Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l(-1) H2O2 and 100 mmol l(-1) Br- at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme.Conclusions: The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions.Significance and Impact of the Study: For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.

Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.

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.

Chemiluminescence studies show that the enzyme vanadium chloroperoxidase from the fungus Curvularia inaequalis is a highly efficient catalyst for the production of singlet oxygen from hydrogen peroxide in a mildly acidic aqueous environment, with a maximum turnover frequency of 100 s 1 with respect to H2O2 consumption. The enzyme studied here can be regarded as a supplementary catalyst to inorganic molybdate, which operates in basic aqueous media. The enzyme system is 10 3 ±1 0 4 times faster than the molybdate system when compared at their optimal pH. The enzyme is very stable against singlet oxygen, in contrast to heme-containing peroxidases which, due to inactivation by singlet oxygen, only produce a short burst, presumably by oxidation of the labile cofactor. The vanadium chloroperoxidase was compared with vanadium bromoperoxidase from the seaweed Ascophyllum nodosum and was found to be even more stable; it is stable towards a continuous flow of singlet oxygen for at least 1 hour and also it is not inactivated by 500 mM H2O2. An important difference with vanadium bromoperoxidase is that the vanadium chloroperoxidase is able to effectively use chloride as a cocatalyst (20 s 1 ) instead of bromide. This offers an important advantage from an application point of view; using chloride, no side products are observed when the chemical trap anthracene-9,10-bis(ethanesulphonate) is fully con- verted by singlet oxygen into its corresponding endoperoxide. By contrast, when bromide is used, 20% side product is formed. During the conversion the enzyme remains fully stable for 25,000 turnovers.

The vanadium-​dependent chloroperoxidase from Curvularia inaequalis is an efficient biocatalyst for the in situ generation of hypohalous acids and subsequent electrophilic oxidn.​/halogenation reactions. Esp., its superb activity and stability under operational conditions make it an attractive catalyst for org. synthesis. Herein, the efficient bromination of thymol was investigated, and turnover nos. of the enzyme were found to exceed 2 000 000. The major novelty of the work is that vanadium chloroperoxidase is more useful as a brominating enzyme than vanadium bromoperoxidase in terms of operational stability, besides being far more stable than heme-​contg. peroxidases.

Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics.

The vanadate cofactor in vanadium chloroperoxidase has been studied using UV-VIS absorption spectroscopy. A band is present in the near-UV that is red-shifted as compared to free vanadate and shifts in both position and intensity upon change in pH. Mutation of vanadate binding residues has a clear effect on the spectrum. Substrate-induced spectral effects allow direct measurement of separate kinetics steps for the first time for vanadium haloperoxidases. A peroxo intermediate is formed upon addition of H(2)O(2), which causes a decrease in the absorption spectrum at 315 nm, as well as an increase at 384 nm. This peroxo form is very stable at pH 8.3, whereas it is less stable at pH 5.0, which is the optimal pH for activity. Upon addition of halides to the peroxo form, the native spectrum is re-formed as a result of halide oxidation. Stopped-flow experiments show that H(2)O(2) binding and Cl(-) oxidation occur on the millisecond to second time scale. These data suggest that the oxidation of Cl(-) to HOCl occurs in at least two steps. In the presence of H(2)O(2), the affinity for the vanadate cofactor was found to be much higher than previously reported for vanadate in the absence of H(2)O(2). This is attributed to the uptake of pervanadate by the apo-enzyme. Human glucose-6-phosphatase, which is evolutionarily related to vanadium chloroperoxidase, is also likely to have a higher affinity for pervanadate than vanadate. This could explain the enhanced insulin mimetic effect of pervanadate as compared to vanadate.

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11 A resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.

Here we describe the isolation, purification, and basic kinetic parameters of a vanadium type chloroperoxidase from the hyphomycete fungus Embellisia didymospora. The enzyme proved to possess similar high substrate affinities, a Km of 5 microM for a bromide, 1.2 mM for a chloride, and 60 microM for a hydrogen peroxide, as those of the vanadium chloroperoxidase from Curvularia inaequalis, although with lower turnover rates for both Cl- and Br-. Substrate bromide was also found to inhibit the enzyme, a feature subsequently also noted for the chloroperoxidase from C. inaequalis. The gene encoding this enzyme was identified using DNA Southern blotting techniques and subsequently isolated and sequenced. A comparison is made between this vanadium chloroperoxidase and that of the fungus C. inaequalis both kinetically and at the sequence level. At the primary structural level the two chloroperoxidases share 68% identity, with conservation of all active site residues.

The optical absorbance spectrum of reduced myeloperoxidase shows an unusual red-shifted Soret band at 472 nm and an an α band at 636 nm. It has been speculated that this red-shift is due to interaction of the protein matrix with the chromophore. The carboxylate side chain of Glu242 is in close proximity of the prosthetic group of the enzyme, and we have examined the effect of the Glu242 to Gin mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation shifts the Soret band in the optical absorption spectrum of the reduced mutated enzyme from 472 to 458 nm. The EPR spectrum was hardly affected and was typical of a rhombic high-spin system (gx = 6.6, gy = 5.2). The alkaline pyridine hemochrome spectrum of the mutant was nearly identical to that of native myeloperoxidase. The resonance Raman spectrum, however, was drastically affected in the mutant. The symmetry-reducing effects were lifted by the mutation and the resonance Raman spectrum was indicative of an iron-porphyrin-like chromophore with a singlet va line at 1367 cm-1. The mutant enzyme was not able to peroxidize chloride to hypochlorous acid. We conclude that the interaction of residue Glu242 with the prosthetic group in native myeloperoxidase is partly responsible for the red-shifted Soret band in the optical spectrum and that this interaction is the origin of the symmetry-reducing effects in the resonance Raman spectrum of the native enzyme. This residue also plays a pivotal role in the ability of the enzyme to peroxidize chloride.

In this article, the role of vanadium in a number of biological systems will be discussed. The metal oxide (vanadate) is present as the prosthetic group in the haloperoxidases, enzymes that are able to oxidize halides in the presence of hydrogen peroxide to hypohalous acids. The bromoperoxidases seem to be involved in the production of the huge amounts of brominated compounds formed by several seaweeds. The enzymes have unusual properties in terms of stability and structural organization. X-ray structures at high resolution are available now for four vanadium haloperoxidases and details of the events occurring at the active site during catalysis are known. The metal oxide binds peroxide in a side-on fashion and seems to acts as a Lewis acid in polarizing the bound peroxide for further nucleophilic attack by an incoming halide. These enzymes are also able to mediate in the presence of hydrogen peroxide, the enantioselective sulfoxidation of organic sulfides yielding chiral sulfoxides with high enantiomeric excess. The active site of the haloperoxidases is very similar to that found in acid phosphatases that hydrolyze phosphate monoesters and there is some evidence that the haloperoxidases have evolved from the phosphatases. Vanadium is found in the vanadiumIII state in certain blood cells of tunicates (Ascidians) under very acidic conditions. Despite many studies, there is no clue as to the physiological role of the metal and there are many questions regarding the mechanism of accumulation and reduction of the metal in these organisms. The metal also seems to be used as an electron sink by certain bacteria under anaerobic conditions. Furthermore, vanadium is accumulated in Amanita species. These toadstools contain up to 10 mM of a vanadiumIV compound with very special structure in which the vanadiumIV is eightfold coordinated in an unusual geometry. Although the compound has been reported to carry out redox reactions, the physiological function is elusive. Keywords: vanadium bromoperoxidase; vanadium chloroperoxidase; X-ray structures; active site; sulfoxidation reactions; tunicates; terminal electron acceptor; amavadine

Previously it has been shown that glycerol can be regioselectively glucosylated by sucrose phosphorylase from Leuconostoc mesenteroides to form 2-O-α- d -glucopyranosyl-glycerol (Goedl et al., Angew. Chem. Int. Ed. 47 (2008) 10086–10089). A series of compounds related to glycerol were investigated by us to determine the scope of the α-glucosylation reaction of sucrose phosphorylase. Both sucrose and glucose 1-phosphate (G1P) were applied as glucosyl donor. Mono-alcohols were not accepted as substrates but several 1,2-diols were readily glucosylated, proving that the vicinal diol unit is crucial for activity. The smallest substrate that was accepted for glucosylation appeared to be ethylene glycol, which was converted to the monoglucoside for 69%. Using high acceptor and donor concentrations (up to 2.5 M), sucrose or G1P hydrolysis (with H2O being the ‘acceptor’) can be minimised. In the study cited above, a preference for glucosylation of glycerol on the 2-position has been observed. For 1,2-propanediol however, the regiochemistry appeared to be dependent on the configuration of the substrate. The (R)-enantiomer was preferentialy glucosylated on its 1-position (ratio 2.5:1), whereas the 2-glucoside is the major product for (S)-1,2-propanediol (1:4.1). d.e.ps of 71–83% were observed with a preference for the (S)-enantiomer of the glucosides of 1,2-propanediol and 1,2-butanediol and the (R)-enantiomer of the glucoside of 3-methoxy-1,2-propanediol. This is the first example of stereoselective glucosylation of a non-natural substrate by sucrose phosphorylase. 3-Amino-1,2-propanediol, 3-chloro-1,2-propanediol, 1-thioglycerol and glyceraldehyde were not accepted as substrates. Generally, the glucoside yield is higher when sucrose is used as a donor rather than G1P, due to the fact that the released phosphate is a stronger inhibitor of the enzyme (in case of G1P) than the released fructose (in case of sucrose). Essentially the same results are obtained with sucrose phosphorylase from Bifidobacterium adolescentis.

Non-ionic microemulsions compatible with the enzyme vanadium chloroperoxidase were designed to perform singlet oxygenation of apolar substrates. The media were based on mono- and polydisperse ethoxylated fatty alcohols (C i E j ), octane and aqueous buffer. “Fish” diagrams were determined to identify the Winsor-boundaries and to formulate a monophasic Winsor IV microemulsion with a minimal surfactant concentration, ensuring less singlet oxygen ( 1 O 2 ) loss than in an aqueous system, thus creating a high oxygenation efficiency. The enzyme was shown to be fully stable in the microemulsion for at least 10 h, converting H 2 O 2 into a constant flow of 1 O 2 in the aqueous microdomains. Part of the 1 O 2 diffuses into the organic compartments prior to fast physical deactivation of 1 O 2 by water molecules. In the apolar domains 1 O 2 quantitatively converts the model substrate 9,10-dimethylanthracene into its corresponding endoperoxide. Near-IR chemiluminescence measurements confirm that the 1 O 2 signal in the microemulsion is higher than in simple aqueous buffer. In a well-stirred (water/octane) biphasic system endoperoxide formation is also observed but the conversion rate is much lower, most likely due to stronger physical quenching of 1 O 2 .

The crystal structure of the apo form of vanadium chloroperoxidase from Curvularia inaequalis reacted with para-nitrophenylphosphate was determined at a resolution of 1.5 A. The aim of this study was to solve structural details of the dephosphorylation reaction catalyzed by this enzyme. Since the chloroperoxidase is functionally and evolutionary related to several acid phosphatases including human glucose-6-phosphatase and a group of membrane-bound lipid phosphatases, the structure sheds light on the details of the dephosphorylation catalyzed by these enzymes as well. The trapped intermediate found is bound to the active site as a metaphosphate anion PO3-, with its phosphorus atom covalently attached to the Nepsilon2 atom of His496. An apical water molecule is within hydrogen-bonding distance to the phosphorus atom of the metaphosphate, and it is in a perfect position for a nucleophilic attack on the metaphosphate-histidine intermediate to form the inorganic phosphate. This is, to our knowledge, the first structural characterization of a real reaction intermediate of the inorganic phosphate group release in a dephosphorylation reaction.

We report 51V solid-state NMR spectroscopy of the 67.5-kDa vanadium chloroperoxidase, at 14.1 T. We demonstrate that, despite the low concentration of vanadium sites in the protein (one per molecule, 1 mumol of vanadium spins in the entire sample), the spinning sideband manifold spanning the central and the satellite transitions is readily detectable. The quadrupolar and chemical shift anisotropy tensors have been determined by numerical simulations of the spinning sideband envelopes and the line shapes of the individual spinning sidebands corresponding to the central transition. The observed quadrupolar coupling constant C(Q) of 10.5 +/- 1.5 MHz and chemical shift anisotropy delta(sigma) of -520 +/- 13 ppm are sensitive reporters of the geometric and electronic structure of the vanadium center. Density functional theory calculations of the NMR spectroscopic observables for an extensive series of active site models indicate that the vanadate cofactor is most likely anionic with one axial hydroxo- group and an equatorial plane consisting of one hydroxo- and two oxo- groups. The work reported in this manuscript is the first example of 51V solid-state NMR spectroscopy applied to probe the vanadium center in a protein directly. This approach yields the detailed coordination environment of the metal unavailable from other experimental measurements and is expected to be generally applicable for studies of diamagnetic vanadium sites in metalloproteins.

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.

We show here that the amino acid residues contributing to the active sites of the vanadate containing haloperoxidases are conserved within three families of acid phosphatases; this suggests that the active sites of these enzymes are very similar. This is confirmed by activity measurements showing that apochloroperoxidase exhibits phosphatase activity. These observations not only reveal interesting evolutionary relationships between these groups of enzymes but may also have important implications for the research on acid phosphatases, especially glucose-6-phosphatase—the enzyme affected in von Gierke disease—of which the predicted membrane topology may have to be reconsidered.

Peroxygenases offer an attractive means to address challenges in selective oxyfunctionalization chemistry. Despite this, their application in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant H2O2. Often atom-inefficient peroxide generation systems are required, which show little potential for large-scale implementation. Here, we show that visible-light-driven, catalytic water oxidation can be used for in situ generation of H2O2 from water, rendering the peroxygenase catalytically active. In this way, the stereoselective oxyfunctionalization of hydrocarbons can be achieved by simply using the catalytic system, water and visible light., Financial support by the European Research Council (ERC Consolidator grant no. 648026) is gratefully acknowledged. The authors thank B. Norder for XRD, W. H. Evers for TEM and F. Hagen for EPR measurements. The authors also thank S. Schmidt for the preparation of benzaldehyde lyase, M. Pesic for the preparation of YqjM and T. Knaus for the preparation of ω-transaminases. F.G.M. received funding as an ERC Starting Grant Fellow (grant agreement 638271).