CLONING AND EXPRESSION OF THE GENE FOR A VANADIUM-DEPENDENT BROMOPEROXIDASE FROM A MARINE MACRO-ALGA, CORALLINA-PILULIFERA

The cDNAs for a vanadium-dependent bromoperoxidase were cloned from a marine macro-alga, Covallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated m olecular mass of 65312 Da in good agreement with that of 64 kDa determ ined for the native enzyme, The deduced amino acid sequence coincided web with partial sequences of peptide fragments of the enzyme. From th e same cDNA library we also isolated another cDNA clone (bpo2) encodin g a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilul ifera growing in a relatively narrow area. The carboxy-terminal 123 re sidues of the enzyme (BPO1) showed an identity of 45% to that of the m arine macroalga Ascophillum nodosum. The homology search of the sequen ces of bromoperoxidases from C, pilulifera (this study) and A. nodosum , and chloroperoxidase from the fungus Curvalaria inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furtherm ore, it was found that the histidine residue directly bound to vanadiu m, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C, inaeq ualis are conserved in the primary structure of the bromoperoxidase fr om C, pilulifera. The cloned bpo1 was introduced into Escherichia coli , and the expressed BPO1 was purified from the recombinant strain. The N-terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N-terminal methionine. ( C) 1998 Federation of European Biochemical Societies.