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Modification of halogen specificity of a vanadium-dependent bromoperoxidase

¹ Department of Biotechnology, Tottori University, Tottori, 680-8552, Japan
² Exeter Biocatalysis Centre, School of Biological and Chemical Sciences, University of Exeter, Exeter EX4 4QD, UK

Reprint request to: Jennifer Littlechild, Exeter Biocatalysis Centre, School of Biological and Chemical Sciences, University of Exeter, Exeter EX4 4QD, UK; e-mail: J.A.Littlechild{at}exeter.ac.uk; fax: 44-1392-263434.

(RECEIVED November 3, 2003; FINAL REVISION January 30, 2004; ACCEPTED February 26, 2004)

	Abstract

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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 NaN₃. 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.

Keywords: haloperoxidase; chloroperoxidase; marine algae; site-directed mutagenesis; vanadate

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03496004.

	Introduction

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Haloperoxidases catalyze the halogenation of organic substrates in the presence of hydrogen peroxide. A variety of halogenated compounds are found in many marine macroalgae. Haloperoxidase enzymes have been characterized from Corallina species (Itoh et al. 1986; Sheffield et al. 1993), Ascophyllum nodosum (Wever et al. 1985), and Ulvella lens (Ohshiro et al. 1999). These enzymes are thought to function in the biosynthesis of halogenated natural products. Haloperoxidases are named after the most electronegative halide they can use; that is, bromoperoxidase (BPO) utilizes Br^– as a substrate, whereas chloroperoxidase (CPO) acts on Cl^–, Br^–, and I^–. Among the marine macroalgae, BPO from the red alga, Corallina pilulifera, has been extensively characterized (Itoh et al. 1985Itoh et al. 1986,1987a,b), and was found to require vanadium in the form of vanadate as an essential cofactor for the enzyme activity (Krenn et al. 1989). The cDNAs for two distinct BPO isomers have been cloned from C. pilulifera (Shimonishi et al. 1998). They shared about 90% homology, and one of them (bpo1) coding for a protein of 598 amino acids with a calculated molecular mass of 65,312 Daltons has been expressed in Escherichia coli and Saccharomyces cerevisiae (Ohshiro et al. 2002). The BPO from Corallina officinalis is similar to the C. pilulifera BPO1 with 92.1% sequence identity. The X-ray structures of both enzymes have been determined (Brindley et al. 1998; Isupov et al. 2000; Littlechild and Garcia-Rodriguez 2003) and they can be superimposed with a 0.5 Å RMS deviation for 593 matching C atoms. Other vanadium-dependent BPOs, for example, from the algae, A. nodosum (de Boer et al. 1986), Fucus distichus (Vreeland et al. 1998), and Laminaria digitata (Colin et al. 2003), and CPOs from the fungi, Curvularia inaequalis (van Schijndel et al. 1993; Simons et al. 1995) and Embellisia didymospora (Barnett et al. 1998) have also been described. The amino acid sequence identities between vanadium BPOs and CPOs are low; however, the amino acids involved in vanadate coordination are highly conserved. The analyses of the three-dimensional structures of the enzymes from A. nodosum (Weyand et al. 1999), C. inaequalis (Messerschmidt and Wever 1996; Macedo-Ribeiro et al. 1999), C. officinalis (Isupov et al. 2000), and C. pilulifera (Littlechild and Garcia-Rodriguez 2003) have confirmed that these residues are in a similar structural arrangement. Because the catalytic site of BPO resembles that of CPO, it is thought that subtle differences in surrounding amino acid residues must determine the halide specificity.

Several mutant haloperoxidase enzymes from C. inaequalis and C. officinalis have been constructed by site-directed mutagenesis (Hemrika et al. 1999; Carter et al. 2002) and their enzymological properties have been examined. In each case the target amino acid residues were located within the active sites, and all the mutant enzymes prepared had lower activity than the wild-type enzyme. This paper describes the identification of two mutant C. pilulifera BPO enzymes that have significant CPO activity. In this study, we demonstrate that a single amino acid substitution can broaden the halogen specificity of this enzyme.

	Results

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Enzyme activity of mutant enzymes
Comparison of the amino acid residues around the catalytic sites of the algal BPO from C. officinalis and the fungal CPOs from C. inaequalis and E. didymospora revealed that the amino acid residue at the position 395 of BPO from C. officinalis, which is close to the catalytic vanadate, is arginine, whereas it is tryptophan in the case of the two fungal CPOs. It was demonstrated that there was no difference in the active sites between two BPOs from C. officinalis and C. pilulifera (Littlechild and Garcia-Rodriguez 2003). We have targeted the corresponding residue of C. pilulifera, R397 (Fig. 1), which has been changed to each of the other 19 amino acid residues by site directed mutagenesis.

View larger version (34K): [in this window] [in a new window]   Figure 1. A diagrammatic representation of the structure and active site of the BPO subunit from C. pilulifera (PDB code 1UP8 [PDB] ). The active site residues are shown as ball-and-stick models. The residues that are conserved in all vanadium BPOs and CPOs are shown in gray. The residues that vary between the different enzymes are shown in cyan. The figure was prepared using the program BOBSCRIPT (Esnouf 1997) and rendered using the program Raster3D (Merritt and Bacon 1997).

View larger version (22K): [in this window] [in a new window]   Figure 2. A histogram representation of the BPO (open bar) and CPO (closed bar) activities of the mutant enzymes in the cell-free extracts after preincubation with 10 mM Na3VO4 at 30°C for 2 h as described in Materials and Methods.

The kinetic studies were performed with the mutant enzymes using KBr, KCl, and hydrogen peroxide as substrates. The results are summarized in Table 1. The K_m values of R397W and R397F for KCl were determined to be 780 mM and 670 mM, respectively (Fig. 3). For the wild-type enzyme, the K_m value for KCl could not be determined because the activity was so low as shown in Figure 3. The addition of 1 M sodium sulfate to the assay mixture had little effect on the rate of the enzyme reaction (data not shown). The K_m values for KBr and hydrogen peroxide were similar for the wild-type and the mutant enzymes. Other enzymatic properties, such as the optimal temperature and pH, heat, and pH stabilities, and the effect of inhibitors on the activities, were also similar for each enzyme except for the inhibitory effect of NaN₃. The activity of the wild-type enzyme was not inhibited by 1 mM NaN₃, while both mutant enzymes lost about 80% of their activities in the presence of 1 mM NaN₃ under the same experimental conditions. It has been demonstrated that the mutant proteins, R397W and R397F, were folded into the same oligomeric state as the wild-type enzyme due to the similar elution profile after gel filtration. Because the other mutant enzymes constructed in this study were not purified, we cannot exclude the possibility that they could have modifications in their folding.

View larger version (19K): [in this window] [in a new window]   Figure 3. The effect of the KCl concentration on the CPO activities of the wild-type enzyme (filled squares), and mutant enzymes R397W (filled circles), and R397F (filled triangles). The reaction mixture contained 100 mM MES buffer (pH 6.5), 60 µM MCD, 2 mM H2O2, the indicated concentrations of KCl and the enzyme was preincubated with vanadate.

	Discussion

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This paper describes the first example of mutant vanadium-dependent BPO enzymes showing significant CPO activity. All of the vanadium-dependent haloperoxidases reported so far have similar structures around their vanadate binding sites and most of the corresponding amino acid residues are conserved. From the protein structure data of BPO from C. officinalis (PDB code 1QHB [PDB] ) and from C. pilulifera (PDB code 1UP8 [PDB] ), the residues shown in Figure 1 surrounding the active site are in identical positions. Among these nine residues, it was proposed that H553 was covalently linked to vanadate, and K400, R408, H487, and R547 (C. pilulifera numbering) formed hydrogen bonds with vanadate oxygens (Littlechild and Garcia-Rodriguez 2003). These five residues and S485 and G486 were conserved in all the haloperoxidases whose amino acid sequences have been determined. The two residues, R397 and H480, vary between the vanadium haloperoxidases from different species. In this study, we have demonstrated that the single amino acid substitution at R397 in BPO from C. pilulifera has changed the substrate specificity for the halogen substrate. However, the CPO activities of the mutant enzymes in the present study were smaller than that of haloperoxidase from the fungi, C. inaequalis (kcat/K_m = 3.4 x 10⁴ M^–1 sec^–1; van Schijndel et al. 1993), which was originally designated as CPO. Additional mutations should be performed to enhance CPO activity of the BPO from C. pilulifera.

We have prepared another mutant enzyme, H480F; however, no enzymatic activity was detected for this protein (data not shown). It has been stated that the biochemical difference between BPO and CPO in the oxidation of halide ions could be attributed to this His residue in BPO from C. officinalis (Isupov et al. 2000) because it is located at the position of the Phe residue, which was suggested to bind the chloride ion in the active site of CPO from C. inaequalis (Messerschmidt and Wever 1996). Our data show that the loss of the activity of the mutant enzyme H480F does not agree with the above proposal; however, we cannot exclude the possibility that this mutant enzyme is misfolded.

It appears that the halogen specificity of the vanadium peroxidases is determined by the affinity of its active site to a particular halide. The results obtained from the mutant enzymes described in this study indicate that the amino acid residues phenylalanine and tryptophan at position 397 favor chloride binding. The location of residue 397 is close to the vanadate binding site in C. pilulifera BPO. It would appear that aromatic amino acid residues favor chloride binding as seen by two tryptophan residues being involved in chloride binding of haloalkane dehalogenase from Xanthobacter autotrophilicus (Verschueren et al. 1993), and a phenylalanine residue located at the chlorine binding site of various amylases (Machius et al. 1995). In the case of BPO from C. pilulifera, the substituted tryptophan or phenylalanine residues at position 397 could participate in chloride binding. In the native BPO enzyme the active site cavity provides the correct electrostatic environment to favor bromide binding to arginine at this site. The corresponding amino acid residue is Trp in BPO from the brown alga, A. nodosum as well as the vanadium CPO. It was reported that this BPO showed CPO activity (Soedjak and Butler 1990), and the value of K_m for KCl was 344 mM, which is the same order of magnitude as those of mutant enzymes in the present study. Therefore, the Trp residue would be preferable for the chloride binding as described above. However, the specific CPO activity of the enzyme from A. nodosum (0.49 U/mg) was much less than those of the mutant enzymes. In the case of the Corallina BPO mutants, which utilize chloride as a substrate, other parts of the protein molecule might play an important role in the progress of the enzyme reaction.

The lack of activity of the R397G, R397A, R397P, and R397S mutants is proposed to be due to the small size of their amino acids side chains. Although the electrostatic potential in the active site favors the halogen binding in these mutants, its binding site is not well defined. Residues such as R397Q, R397L, and R397E will aid solvent exclusion and restrict the position of the halide ion to the correct site for halogenation. The reason why the R397Y mutant enzyme shows no CPO activity, in contrast to the other two aromatic mutant enzymes R397W and R397F, could be due to its deprotonation in the environment of the active site. The bulky tryptophan residue of the R397W mutant enzyme also appears to hinder the incorporation of vanadate as it is located at the entrance of vanadate binding pocket.

Regarding the inhibition of the mutant enzymes by azide, it would appear that R397 ensures specific binding of the charged bromide ion and disfavors binding of azide. In the two mutant enzymes R397W and R397F, however, hydrophobic interaction would provide binding for both chloride and bromide. Azide ions also have high affinity for this site and compete for binding, thus inhibiting the halogenation reaction.

The single amino acid substitution of R397 increases the affinity of the BPO enzyme for chloride ions thereby broadening its halide specificity. The structural analysis of the mutant enzymes is in progress, and these results will further our understanding of halogen binding and specificity in the vanadium-dependent haloperoxidases.

	Materials and methods

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Plasmid and microorganism
The plasmid pTNT30, a yeast-E. coli shuttle vector, contained the bpo1 gene of C. pilulifera (Ohshiro et al. 2002). S. cerevisiae strain BJ1991 was used as the host of the expression plasmid. Transformation of the strain BJ1991 was performed according to the method previously reported (Hemrika et al. 1999; Ohshiro et al. 2002).

Site-directed mutagenesis
Site-directed mutagenesis was carried out with the QuickChange site-directed mutagenesis kit (Stratagene) using two oligonucleo-tides per each amino acid replacement according to the manufacturer’s instructions. The mutagenic primers were designed to alter the codon of R397 to those of the other 19 amino acids and the codon of H480 to that of phenylalanine. The DNA sequences of the mutant enzymes were confirmed using an ABI 373 or an ABI PRISM 3100 automated DNA sequencer.

Cultivation of the recombinant yeast
The recombinant yeast was cultivated as described previously (Ohshiro et al. 2002). The preculture was carried out with the medium containing 0.67% Yeast nitrogen base w/o amino acids (Difco), 2% glucose, 40 µg/mL of L-tryptophan, and 50 µg/mL of L-leucine. The precultured broth was inoculated into the production medium containing 2% yeast extract, 2% peptone, and 4% D-galactose. Cultivation was carried out at 30°C for 2 days. To prepare the cell-free extracts of each mutant strain, the cells were suspended in 50 mM Tris-SO₄ buffer (pH 7.4), and disrupted with an ultraoscillator (Sonifier 450; Branson Instruments) with 0.5-mm glass beads for 1 h on ice. The supernatants after the removal of cell debris by the centrifugation were used directly for the enzyme assays.

Purification of the mutant enzymes
The mutant enzymes, R397W and R397F, were purified based upon the method described previously (Ohshiro et al. 2002). All purification procedures were carried out at 4°C or on ice unless otherwise stated. The buffer A, 50 mM Tris-SO₄ buffer (pH 7.4) was used throughout. The suspended cells in this buffer were disrupted with 0.5-mm glass beads through a Dyno-Mill homogeneizer (Willy A. Bachofen). Ammonium sulfate fractionation was carried out with the supernatant (cell-free extract) after the centrifugation as described previously (Ohshiro et al. 2002). The dialyzed enzyme solutions were applied to DEAE-Sepharose columns (5.8 x 30 cm), which had been equilibrated with the same buffer. The columns were washed with the same buffer followed by 0.2 M KCl in buffer A. The mutant enzymes R397W and R397F were eluted with 0.2 M and 0.35 M KCl in buffer A, respectively. The active fractions were combined, concentrated by ultrafiltration, and dialyzed against buffer A. The dialyzed enzyme solutions were applied onto Q-Sepharose columns (1.7 x 15 cm), which had been equilibrated with buffer A. The columns were washed with 0.3 M (R397W) or 0.35 M KCl (R397F) in buffer A, and the bound proteins were eluted with 0.4 M KCl in buffer A. For R397W enzyme, the concentrated enzyme solution was once again applied onto a Q-Sepharose column (1.7 x 11 cm), which had been equilibrated with buffer A. After the column was washed with 0.3 and 0.35 M KCl in buffer A, the enzyme was eluted with 0.4 M KCl in buffer A. For R397F enzyme, the concentrated enzyme solution was applied onto a Sepharose CL-4B column (1.8 x 94 cm), which had been equilibrated with buffer A. The active fractions were combined and concentrated by ultrafiltration. The wild-type enzyme was purified according to the same method as previously described (Ohshiro et al. 2002). The gel filtration step was performed for all enzymes to ensure the homogeneity of the preparation.

Enzyme assays and other analytical methods
The enzyme solution was preincubated with 10 mM Na₃VO₄ at 30°C for 2 h before measuring the enzyme activity. BPO activity was determined spectrophotometrically by the halogenation of monochlorodimedone (MCD, = 19.9 mM^–1 cm^–1 at 290 nm) as described previously (Yamada et al. 1985). The reaction mixture contained 100 mM MES buffer (pH 6.5), 100 KBr, 60 µM MCD, 2 mM H₂O₂, 1 mM Na₃VO₄, and the enzyme. The assay temperature was 30°C and activity followed by decrease in absorbance at 290 nm (Ohshiro et al. 2002). When measuring chloroperoxidase activity, 1.5 M KCl (99.5%, Wako Chemicals) was added to the reaction mixture instead of KBr. The protein concentration was determined by the method of Bradford (1976) using a Bio-Rad protein assay reagent with bovine serum albumin as a standard. SDS-PAGE was carried out by the method described by Laemmli (1970) employing a 12.5% gel for separation. Protein bands were visualized by staining with Coomassie Brilliant Blue G-250 dissolved in 50% methanol–10% acetic acid and destained in 30% methanol–10% acetic acid.

	Acknowledgments

 
The Biotechnology and Biological Research Council (BBSRC), UK, is thanked for financial support for a postdoctoral fellowship to M.N.I. and support to E.G.R. The work has been carried out as part of a Japan partnership award from the BBSRC. This work was also supported in part by a Grant-in-Aid for Scientific Research (No.15404025) from the Japan Society for the Promotion of Science.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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