牙鲆碱性磷酸酶cDNA序列分析与蛋白质高级结构预测

为研究碱性磷酸酶(EC 3.1.3.1; alkaline phosphatase，ALP)在牙鲆(Paralichthys Olivaceus)发育和变态中的作用，采用RACE的方法克隆了牙鲆ALP基因cDNA全长，通过生物信息学分析了核苷酸序列并进行蛋白结构预测. 结果表明，牙鲆ALP cDNA全长为1 811bp，能编码476个氨基酸的蛋白质，分子量为52 293.1，等电点为7.67. 编码区核苷酸GC含量在ALP同源基因中差异比较大，脊椎动物明显高于非脊椎动物和细菌. 分子系统分析显示，牙鲆ALP和青黑斑河豚(Tetraodon nigroviridis)、斑马鱼（Danio rerio）的组织非特异性ALP有较高的同源性，分子进化树和物种进化树是一致的. 在蛋白序列中的一些重要的功能位点，包括金属离子结合位点、N糖基化位点和丝氨酸磷酸化位点等表现了较高的保守性. 牙鲆ALP和人胎盘ALP(PALP)在蛋白序列上有43%的相似性，其3D结构非常接近.通过氨基酸空间位置比较发现，牙鲆ALP中141和203位半胱氨酸对应于人PALP的121和183位半胱氨酸，推测能形成一个二硫键. 在两者酶活性中心，3个金属离子结合的氨基酸残基非常保守，Zn离子周围的9个氨基酸中有2个不同；Mg离子周围的7个氨基酸也只有2个不同，包括一对类似的丝氨酸155和苏氨酸175.

cDNA Sequence Analysis and Tertiary Structure Prediction of Alkaline Phosphatase from Paralichthys olivaceus

To understand the function of alkaline phosphatase(ALP)in the development and metamorphosis of Paralichthys Olivaceus,full-length cDNA of ALP was cloned by the technique of RACE.The cDNA and deduced protein sequence were analyzed by bioinformatics.The results show that the full-length cDNA sequence consists of 1 811 bp,including an ORF encoding for 476 amino acids and a terminator codon.The GC content of coding region is different in selected ALPs.The predicted molecular weight of the deduced protein is 52 293.1 and the pI is 7.67.Molecular phylogeny tree shows that the ALP of Paralichthys Olivaceus shares high homology with the ALP of Tetraodon nigroviridis and Danio rerio.Several ALP functional sites,including the metal ion binding sites,phosphoserine intermediate,N-linked glycosylation sites,are conservative in those selected sequences.On tertiary structure prediction and structure alignment with human PALP,the flounder ALP has high similarity with that of human PALP,while 43% similarity is shared by the primary structure of them.It might possess one disulfide bond,which is composed by the cysteines 141 and 203.Few of the amino acids residues around the Zn and Mg metal ions are different between the above mentioned two kinds of ALP,including two similar amino acids,i.e.S155,T175.