Crystal Structure of a Trapped Phosphate Intermediate in Vanadium Apochloroperoxidase Catalyzing a Dephosphorylation Reaction ‡ Sandra de Macedo-Ribeiro, §,| Rokus Renirie, ⊥ Ron Wever, ⊥ and Albrecht Messerschmidt* ,§ Abteilung Proteomics und Signaltransduktion, Max-Planck-Institut fu ¨r Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany, and Van’t Hoff Institute of Molecular Sciences, UniVersity of Amsterdam, 1018 WS Amsterdam, The Netherlands ReceiVed September 12, 2007; ReVised Manuscript ReceiVed NoVember 14, 2007 ABSTRACT: 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 Å. 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 PO 3 - , with its phosphorus atom covalently attached to the N ǫ2 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 dephospho- rylation reaction. Vanadium haloperoxidases are enzymes that catalyze the oxidation of halides to their corresponding hypohalous acids at the expense of hydrogen peroxide Haloperoxidases are named after the most electronegative halide they are able to oxidize; thus, a chloroperoxidase oxidizes Cl - , Br - , and I - . If a convenient nucleophilic acceptor is present, a reaction will occur with HOX to form a diversity of halogenated reaction products. Many of these organohalogens have biocidal effects, and this may provide defense functions. The crystal structure of the first vanadium haloperoxidase, the chloroperoxidase (VCPO) 1 from the fungus CurVularia inaequalis, and the proposal of a mechanism based on the structure, mutation studies, and functional analyses allowed a further understanding of the chemistry of vanadium in this system (1-5). VCPO is related to vanadium bromoperoxi- dases (VBPOs) from the brown seaweed Ascophyllum nodosum and the red algae Corallina species, whose crystal structures have also been solved (6, 7). The VBPOs show a similar mainly R-helical fold of the monomers, but they share only a common four-helix bundle with VCPO. The amino acid (aa) residue stretch spanning the four-helix bundle and the subsequent C-terminal loop contains all residues forming the active site that binds the vanadium in the form of vanadate (HVO 4 2- ) as the prosthetic group. The structures of the actives site of all vanadium haloperoxidases align very well. The vanadium is coordinated to His496 (VCPO numbering throughout if not otherwise stated), and the residues Lys353, Arg360, Ser402, Gly403, and Arg490 form hydrogen bonds with the non-protein oxygens of vanadate. His404 was proposed to function as the acid-base group in catalysis. The main difference between VCPOs and VBPOs is the substitution of Phe397 to His (411 in VBPO from A. nodosum). An aa sequence database search revealed an unexpected homology between the vanadium haloperoxidases and three families of acid phosphatases, indicating similarities in the anion binding active sites of all these enzymes including human glucose-6-phosphatase (8-10) and lipid phosphate hydrolases (11). Together with the similar structural and chemical features of vanadate and phosphate, this prompted a study to check the possible phosphatase activity of vanadium haloperoxidases. Indeed, the recombinant apo- VCPO using para-nitrophenylphosphate (pNPP) as a sub- strate showed phosphatase activity (8). Similarly, acid phosphatase substituted with vanadate in the active site showed haloperoxidase activity (12). The crystal structures of the acid phosphatases from Escherichia blattae (EB-AP) and Salmonella typhimurium (PhoN), members of the family related to the haloperoxi- dases, were determined both in the native form with sulfate ‡ Coordinates of the structure described in this paper have been deposited in the Protein Data Bank with ID 3BB0. * Corresponding author. Tel.: +49 89 8578 2669; fax: +49 89 8578 2219; e-mail: messersc@biochem.mpg.de. § Max-Planck-Institut fu ¨r Biochemie. | Current address: Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, 4150-180 Porto, Portugal. ⊥ University of Amsterdam. 1 Abbreviations: VCPO, vanadium chloroperoxidase from CurVu- laria inaequalis; VBPO, vanadium bromoperoxidase; pNPP, para- nitrophenylphosphate; EB-AP, acid phosphatase from Escherichia blattae; PhoN, acid phosphatase from Salmonella typhimurium; apo- VCPO-pNPP, apo-VCPO reacted with para-nitrophenylphosphate; Kd, dissociation constant; aa, amino acid; rmsd, root mean square deviation; ESU, estimated standard uncertainty. H 2 O 2 + H + + X - f H 2 O + HOX (1) 929 Biochemistry 2008, 47, 929-934 10.1021/bi7018628 CCC: $40.75 © 2008 American Chemical Society Published on Web 12/29/2007