doi:10.1006/jmbi.2000.3806availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (2000) 299, 1035±1049 Crystal Structure of Dodecameric Vanadium- dependent Bromoperoxidase from the Red Algae Corallina officinalis MichailN.Isupov1,AndrewR.Dalby1,AmandaA.Brindley1 YoshikazuIzumi2,TadashiTanabe3,GaribN.Murshudov4,5 andJenniferA.Littlechild1* 1Schools of Chemistry and The three-dimensional structure of the vanadium bromoperoxidase pro- Biological Sciences, University tein from the marine red macroalgae Corallina of®cinalis has been deter- of Exeter, Stocker Road, Exeter mined by single isomorphous replacement at 2.3 AÊ resolution. The EX4 4QD, UK enzyme subunit is made up of 595 amino acid residues folded into a 2 single a b domain. There are 12 bromoperoxidase subunits, arranged Department of Biotechnology with 23-point group symmetry. A cavity is formed by the N terminus of Faculty of Engineering, Tottori each subunit in the centre of the dodecamer. The subunit fold and dimer University, Tottori, Koyama- organisation of the Cor. of®cinalis vanadium bromoperoxidase are similar cho 980, Japan to those of the dimeric enzyme from the brown algae Ascophyllum nodo- 3Department of Pharmacology sum, with which it shares 33 % sequence identity. The different oligomeric National Cardiovascular Centre state of the two algal enzymes seems to re¯ect separate mechanisms of Research Institute, Fujishiro- adaptation to harsh environmental conditions and/or to chemically day, Suita, 565-8565, Japan active substrates and products. The residues involved in the vanadate 4 binding are conserved between the two algal bromoperoxidases and the Department of Chemistry vanadium chloroperoxidase from the fungus Curvularia inaequalis. University of York, Heslington However, most of the other residues forming the active-site cavity are York, Y01 5DD, UK different in the three enzymes, which re¯ects differences in the substrate 5CLRC, Daresbury Laboratory speci®city and stereoselectivity of the reaction. A dimer of the Daresbury, Warrington Cor. of®cinalis enzyme partially superimposes with the two-domain WA4 4AD, UK monomer of the fungal enzyme. # 2000 Academic Press Keywords: vanadium; haloperoxidase; dodecamer; enzyme structure; *Corresponding author vanadium binding site Introduction halide to nucleophilic organic substrates in the pre- sence of hydrogen peroxide is catalysed by halo- Many naturally occurring halogenated organic peroxidases. These enzymes have been isolated compounds have been isolated; this list is con- fromadiverserangeoforganisms(Ahernetal., stantlyincreasing(Gribble,1992).Theyarepro- 1980;Manthey&Hager,1981;VanPeeÂ&Lingens, duced by seaweeds, sponges, lichens and fungi 1985;Zeng&Fenna,1992;Itohetal.,1994).Halo- and are thought to be associated with defence peroxidases are named according to the most elec- mechanisms preventing the organism from being tronegativehalogentheyoxidise(Hemrikaetal., eaten by predators and to aid colonisation in natu- 1997).Onthebasisoftheircofactorrequirement ralhabitats(Simonsetal.,1995).Theadditionof they are classi®ed into the three groups: heme- containing, vanadium-containing and metal-free haloperoxidases(Hemrikaetal.,1997). Abbreviations used: VBPO, vanadium A bromoperoxidase enzyme with an absolute bromoperoxidase; AVBPO, Ascophyllum nodosum VBPO; requirement for vanadium (VBPO) was ®rst iso- CVBPO, Corallina of®cinalis VBPO; H-bond, hydrogen lated from the brown macro-algae Ascophyllum bond; NCS, non-crystallographic symmetry; F , o nodosum(Weveretal.,1985).Onemoleculeofvana- observed structure factors; Fc, calculated structure factors. date per subunit was shown to be required for the E-mail address of the corresponding author: enzymeactivity(Weveretal.,1988).EPRstudies [email protected] (Weveretal.,1988)andK-edgeX-rayabsorption 0022-2836/00/041035±15 $35.00/0 # 2000 Academic Press 1036 Vanadium-dependent Bromoperoxidase studies(Kusthardtetal.,1993)onthebromoperoxi- suggested that the bromide ion does not coordinate dase from A. nodosum (AVBPO) showed that the to the vanadium centre, but is bound covalently to oxidation state of the metal was vanadium V. The acarbonatom(Dauetal.,1999). redox state of the metal is not thought to change The VBPO from the red macroalgae Cor. of®cina- during turnover of the enzyme. It has been pro- lis (CVBPO), a seaweed found on the south Devon posed that the function of the vanadium is to bind coast, has been isolated and characterised hydrogen peroxide to yield an activated peroxo (Shef®eldetal.,1993).Itwassuggestedtobea intermediate, which is able to react with bromide homododecamer based on its subunit molecular toproduceHOBr(Weveretal.,1988).Vanadium- mass of 64 kDa from SDS-gel electrophoresis, and dependenthaloperoxidaseenzymeshavenow a total molecular mass from equilibrium centrifu- beenisolatedfromanumberofmarinealgae gationstudiesof740kDa(Shef®eldetal.,1993). (Weveretal.,1985;Itohetal.,1986;Shef®eldetal., Itohetal.(1986)reportedthesamesubunitmol- 1993)andalsofromsomelichensandfungi(Van ecular mass and the oligomer molecular mass Schijndeletal.,1993;Platetal.,1987). of 790(Æ20) kDa for the related enzyme from Theprimaryaminoacidsequenceofthe Cor. pilulifera. vanadiumchloroperoxidasesfromthefungal The VBPO enzymes from Corallina species have speciesCurvulariainaequalis(Simonsetal.,1995), been shown to be thermostable, stable to organic Embellisia didymospora (Barnett et al., 1998) and solvents and also resistant to high concentrations Drechslerabiseptata(partialsequence;Hemrikaetal., ofhydrogenperoxide(Shef®eldetal.,1993;Itoh 1997)showsequenceidentityintherangeof65- etal.,1986).ThesefeaturesmakeVBPOacandidate 95 %. The two vanadium bromoperoxidases from for use as an industrial catalyst in biotransform- theredmacroalgaeCorallinapilulifera(Shimonishi ationreactions(Soedjak&Butler,1990;Neidleman etal.,1998)showsequenceidentityof91%andthe &Geigert,1987;Shef®eldetal.,1995).CVBPOhas VBPOsfromthebrownmacroalgaeFucusdistichus been shown to perform regio-speci®c bromination (Vreelandetal.,1998)andA.nodosum(Vilter,1995; of cinnamyl substrates and aromatic heterocycles Weyandetal.,1999)haveasequenceidentityof as well as the stereo-speci®c oxidation of pro-chiral 86 %. The VBPO enzymes from the red algae show sulphideions(Itohetal.,1987,1988;Coughlinetal., 32-34%identitytothosefromthebrownalgae. 1993;Andersonetal.,1997). There is, however, less sequence identity between The enzyme from Cor. of®cinalis has been crystal- the algal VBPO and the fungal vanadium chloro- lisedintwodifferentforms(Rushetal.,1995; peroxidase enzymes. Only the catalytic C-terminal Brindleyetal.,1998).Acubiccrystalformwas regions can be aligned with no more than 26 % grown in the presence of vanadium, and a tetra- identity within a 103 amino acid residue overlap. gonal form was grown from ammonium phos- The acid phosphatases show sequence identity to phate. The phosphate group has been shown to the vanadium-binding site motifs in haloperoxi- compete with vanadate for binding in the enzyme daseenzymes(Hemrikaetal.,1997). activesite(Hemrikaetal.,1997).Preliminarycrys- The X-ray crystallographic structures of the tallographic analysis has shown that the CVBPO chloroperoxidase from the fungus Cur. inaequalis in dodecamer has its subunits arranged with a cubic both the native and peroxide-bound forms have 23-pointgroupsymmetry(Brindleyetal.,1998). beendetermined(Messerschmidt&Wever,1996; WereportheretheCVBPOcrystalstructuredeter- Messerschmidtetal.,1997).Themonomerofthe mined to 2.3 AÊ resolution in its complex with inor- enzyme has two a-helical domains with a similar ganic phosphate and its comparison with the fold, the four a-helix bundles of each domain are AVBPO and the Cur. inaequalis chloroperoxidase related by approximately 180 rotation structures. (Messerschmidt&Wever,1996).Theactivesiteis located at the end of a four-a-helix cluster in the C-terminal domain, the residues of the N-terminal Results and Discussion domain contribute to the active site. Kinetic Quality of the model (Hemrikaetal.,1999)andstructural(Macedo- Ribeiroetal.,1999)studiesofmutantchloroperoxi- The CVBPO structure has been re®ned to an R- dase proteins have recently shown the importance factor of 0.172 for all data in the resolution range of histidine, which forms a chemical bond to vana- 22-2.3 AÊ without a s cut-off. This excluded 2.0 % dateandthreebasicresiduesinvolvedinthevana- of the randomly distributed re¯ections assigned to date-bindingsiteforthischloroperoxidase calculate the Rfree of 0.227. The asymmetric unit reaction. A mechanism for the halogenation reac- contains six subunits. One of the molecular dyads tionwasproposedbyMesserschmidt&Wever of the CVBPO dodecamer coincides with a diag- (1996)andthishasbeendevelopedbyHemrika onal crystallographic 2-fold axis. The electron den- etal.(1999).Thecrystalstructureofthedimeric sity allowed the positioning of all 595 amino acid VBPO from the marine brown algae A. nodosum residues within each subunit. The model also con- (Weyandetal.,1999)wasrecentlyreportedwhich tains 2186 water molecules, six phosphate ions and reinforced the proposed mechanism for chloroper- six magnesium ions. oxidase. Bromine K-edge EXAFS studies used to Thedispersion-precisionindicator(Murshudov investigate the binding of bromide to AVBPO have &Dodson,1997)givesanoverallestimateofroot Vanadium-dependent Bromoperoxidase 1037 mean square error in the co-ordinates of 0.18 AÊ for (Figure1).Outof51sequencedifferencesbetween the well-de®ned part of the structure. The overall the