Shimonishi M, Kuwamoto S, Inoue H, Wever R, Ohshiro T, Izumi Y, Tanabe T
Department of Pharmacology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
FEBS Lett. 1998 May 22;428(1-2):105-10. doi: 10.1016/s0014-5793(98)00500-6.
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.
从海洋大型藻类珊瑚藻中克隆了钒依赖性溴过氧化物酶的cDNA。一个克隆（bpo1）的开放阅读框编码一个598个氨基酸的蛋白质，计算分子量为65312 Da，与天然酶测定的64 kDa分子量非常一致。推导的氨基酸序列与该酶肽片段的部分序列非常吻合。从同一个cDNA文库中，我们还分离出另一个cDNA克隆（bpo2），它编码一个597个氨基酸的蛋白质，与BPO1的同一性约为90%，这表明生长在相对狭窄区域的珊瑚藻溴过氧化物酶基因存在遗传多样性。该酶（BPO1）的羧基末端123个残基与海洋大型藻类鹿角菜的羧基末端123个残基的同一性为45%。对珊瑚藻（本研究）和鹿角菜的溴过氧化物酶序列以及不等弯孢霉菌的氯过氧化物酶序列进行同源性搜索，结果表明存在高度保守的序列PxYxSGHA和LxxxxAxxRxxxGxHxxxD。此外，还发现与钒直接结合的组氨酸残基、构成金属中心的其他残基以及形成不等弯孢霉菌氯过氧化物酶活性位点的催化组氨酸残基在珊瑚藻溴过氧化物酶的一级结构中是保守的。将克隆的hpol导入大肠杆菌，并从重组菌株中纯化表达的PO1。纯化的BPO1的N端氨基酸序列与cDNA推导的序列相同，只是N端的甲硫氨酸除外。
