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, Corallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65312 Da in good agreement with that of 64 kDa determined for the native enzyme. The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme. From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilulifera growing in a relatively narrow area. The carboxy-terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro-alga Ascophillum nodosum. The homology search of the sequences of bromoperoxidases from C. pilulifera (this study) and A. nodostum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C. inaequalis are conserved in the primary structure of the bromoperoxidase from C. pilulifera. The cloned hpol was introduced into Escherichia coli, and the expressed PO1 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.
Shimonishi
M
M
Department of Pharmacology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Kuwamoto
S
S
Inoue
H
H
Wever
R
R
Ohshiro
T
T
Izumi
Y
Y
Tanabe
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism; D000461:Rhodophyta / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014639:Vanadium / Q000378:metabolism*
D014639:Vanadium; D010544:Peroxidases; C026195:bromide peroxidase