Modification of halogen specificity of a vanadium‐dependent bromoperoxidase

Ohshiro, Takashi; Littlechild, Jennifer A.; Garcia-Rodriguez, Esther; Isupov, Michail N.; Iida, Yusuke; Kobayashi, Takushi; Izumi, Yoshikazu

doi:10.1110/ps.03496004

Cited by 33 publications

(19 citation statements)

References 34 publications

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“…a Phe397 in VCPO ( Figure 4B) replacing a histidine residue in VBPO, was responsible for the difference in halogen specificity [66]. Previously, the mutation of the arginine residue (Arg397) in the red algal VBPO from C. pilulifera ( Figure 4F) has conferred significant chloroperoxidase activity to mutant enzymes R397W and R397F [94,107]. This residue is replaced by a tryptophan residue in the fungal VCPO.…”

Section: Vo 4 Coordination In the Crystal Structure Of Vanadium Halopmentioning

confidence: 97%

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

Leblanc

Vilter

Fournier

et al. 2015

Coordination Chemistry Reviews

123

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In the environment, vanadium-dependent haloperoxidases (VHPO) are likely to play a key role in the production of biogenic organo-halogens. These enzymes contain vanadate as a prosthetic group, and catalyze, in the presence of hydrogen peroxide, the oxidation of halide ions (Cl -, Br -or I -). They are classified according to the most electronegative halide that they can oxidize. Since the first discovery of a vanadium bromoperoxidase in the brown alga Ascophyllum nodosum thirty years ago, structural and mechanistic studies have been mainly conducted on two types of VHPO, chloro-and bromoperoxidases, and more recently on a vanadium-dependent iodoperoxidase. In this review, we highlight the main progress obtained on the structure-function relation of these proteins, based on biochemistry, crystallography and X-ray absorption spectroscopy (XAS). The comparison of 3D protein structures of the different VHPO helped identify the residues that govern the molecular mechanisms of catalysis and specificity of VHPO. Vanadium K-edge XAS gave further important insight to understand the fine changes around the vanadium cofactor during the catalytic cycle. The combination of different structural approaches, at different scales of resolution, shed new light on biological vanadium coordination in the active site, and its importance for the catalytic cycle and halide specificity of vanadium haloperoxidases. Keywords (max 6)Vanadium-dependent haloperoxidase, vanadium coordination, crystallographic structure, structural evolution, oligomeric state, X-ray absorption spectroscopy on biological vanadium

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Section: Vo 4 Coordination In the Crystal Structure Of Vanadium Halopmentioning

confidence: 97%

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

Leblanc

Vilter

Fournier

et al. 2015

Coordination Chemistry Reviews

123

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“…The brominating activity of this mutant at pH 5 is increased ϳ6-fold to a k cat of 575 s Ϫ1 , which is one of the highest values for Br Ϫ turnover reported for a vanadium haloperoxidase. The chlorinating activity of the P395D/L241V/T343A mutant is ϳ2-fold higher than the wild-type enzyme, thereby making this the best chlorinating vanadium haloperoxidase (21,22).…”

Section: E Coli Expression System and Generation Ofmentioning

confidence: 99%

“…The assigned full positive charge on the protonated peroxide oxygen in VCPO was also suggested to give rise to the very strong uncompetitive inhibition by azide seen in VCPO, being absent in wild-type VBPOs (20). A VBPO mutant from C. pilulifera that essentially has become a VCPO was also strongly inhibited by azide (21). The His-411 of VBPO from A. nodosum also forms a hydrogen bond with the catalytically important Lys-341, and it was suggested that this decreases the polarizing effect of this histidine on the bound peroxide (15).…”

mentioning

confidence: 99%

Laboratory-evolved Vanadium Chloroperoxidase Exhibits 100-Fold Higher Halogenating Activity at Alkaline pH

Hasan

Renirie

Kerkman³

et al. 2006

Journal of Biological Chemistry

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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 M) 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.Haloperoxidases catalyze the oxidation of halides to hypohalous acids (see Equation 1), an industrially interesting reaction; these enzymes can be used to halogenate various organic compounds (1, 2) (see Equation 2). In addition they may provide an alternative biocide in antifouling applications (3-5) or may be used as a component in disinfectants and in detergent formulations for bleaching purposes (6 -8). Equations 1 and 2 illustrate the reactions occuring.where X is clorine, bromine, or iodine, and A is an organic nucleophilic acceptor.Presently two classes of enzymes are known that efficiently catalyze the oxidation of a halide by hydrogen peroxide, the vanadium haloperoxidases and the heme peroxidases. The heme peroxidases have a significant disadvantage of rapid inactivation during turnover because of an oxidative reaction of the heme group with the peroxide substrate and the formed hypohalous acids. Vanadium haloperoxidases (VHPOs), 2 which contain vanadate (VO 4 3Ϫ ) as a prosthetic group, do not suffer from this disadvantage (9); in addition they are much more resistant toward heat, detergent, and solvent denaturation (10). The major drawback of all haloperoxidases including the stable VHPOs is that they are mainly active at mildly acidic pH values, whereas for many applications activity at mildly alkaline pH values is required. An example of this is their potential use as an antif...

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“…The second step, i.e., the oxidation of the halide, is the determinant for the enzymatic specificity of VHPO but has yet to be elucidated. Targeted active site mutants of C. inaequalis VCPO or C. pilulifera and Gracilaria changii VBPO have shown drastic reductions or increases of chlorinating activity, respectively (20)(21)(22). According to these structure-function studies, the catalytic properties and halide specificities of VCPO and VBPO are likely to be dependent on the electronic environment around the VO 4 moiety (4,15,23).…”

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confidence: 94%

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

Fournier

Rebuffet

Delage

et al. 2014

Appl Environ Microbiol

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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 ␣-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. H alogenated compounds have various biological functions in nature, ranging from chemical defense to signaling. Indeed, halogenation (i.e., iodination, bromination, or chlorination) is an efficient strategy used to increase the biological activity of secondary metabolites and involves many different halogenating enzymes (1-4). Among them, vanadium-dependent haloperoxidases (VHPO) contain the bare metal oxide vanadate as a prosthetic group. In the presence of hydrogen peroxide, VHPO enzymes catalyze the oxidation of halides according to the reaction H 2 O 2 ϩ X Ϫ ϩ H ϩ ¡ H 2 O ϩ HOX, wherein X Ϫ represents a halide ion and may be Cl Ϫ , Br Ϫ , or I Ϫ (4). A variety of halocarbons can subsequently be generated if the appropriate nucleophilic acceptors are present. The nomenclature of vanadium-dependent haloperoxidases is based on the most electronegative halide they can oxidize: chloroperoxidases (VCPO) can catalyze the oxidation of three different halides, i.e., chloride, bromide and iodide; bromoperoxidases (VBPO) can oxidize only bromide and iodide; and iodoperoxidases (VIPO) are specific for iodide.The first VBPO was discovered 30 years ago, in the brown alga Ascophyllum nodosum (5). Since then, structural and mechanistic studies have focused on two types of eukaryotic VHPO, namely, VCPO from the pathogenic fungus Curvularia inaequalis (6) a...

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Modification of halogen specificity of a vanadium‐dependent bromoperoxidase

Cited by 33 publications

References 34 publications

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

Laboratory-evolved Vanadium Chloroperoxidase Exhibits 100-Fold Higher Halogenating Activity at Alkaline pH

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

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