Hatakeyama K, Inoue Y, Harada T, Kagamiyama H
Department of Medical Chemistry, Osaka Medical College, Japan.
J Biol Chem. 1991 Jan 15;266(2):765-9.
A full-length cDNA clone for GTP cyclohydrolase I, the first enzyme of the tetrahydrobiopterin biosynthetic pathway, was isolated and characterized. Synthetic oligonucleotides, constructed according to selected amino acid sequences of purified GTP cyclohydrolase I, were used to screen a rat liver cDNA library. Four clones were isolated, and the length of the longest cDNA insert was 1024 base pairs. The identity of the cDNA was confirmed by amino acid sequence data for eight fragments obtained by lysyl endopeptidase digestion of the purified protein. The coding region encoded a protein of 241 amino acid residues, but the NH2 terminus of the protein contained 11 additional amino acid residues not present in the purified protein. RNA blot analysis showed a single mRNA species of 1.2 kilobases in rat liver. A characteristic feature of the deduced amino acid sequence of GTP cyclohydrolase I was the presence of sequences similar to those proposed for the phosphorylation sites for casein kinase II and growth-associated histone H1 kinase. Furthermore, significant similarity was found to the highly conserved sequences of dihydrofolate reductases, which are known to be involved in the binding of the pterin group of dihydrofolate to the reductases. This region in GTP cyclohydrolase I may be assigned to the binding site of tetrahydrobiopterin, one of the inhibitors of this enzyme.
分离并鉴定了四氢生物蝶呤生物合成途径中首个酶——GTP环化水解酶I的全长cDNA克隆。根据纯化的GTP环化水解酶I的选定氨基酸序列构建的合成寡核苷酸，用于筛选大鼠肝脏cDNA文库。分离出四个克隆，最长的cDNA插入片段长度为1024个碱基对。通过对纯化蛋白进行赖氨酰内肽酶消化获得的八个片段的氨基酸序列数据，证实了该cDNA的身份。编码区编码一个由241个氨基酸残基组成的蛋白质，但该蛋白质的NH2末端含有11个纯化蛋白中不存在的额外氨基酸残基。RNA印迹分析显示大鼠肝脏中有一个1.2千碱基的单一mRNA种类。GTP环化水解酶I推导的氨基酸序列的一个特征是存在与酪蛋白激酶II和生长相关组蛋白H1激酶磷酸化位点相似的序列。此外，发现与二氢叶酸还原酶的高度保守序列有显著相似性，已知二氢叶酸还原酶参与二氢叶酸的蝶呤基团与还原酶的结合。GTP环化水解酶I中的这一区域可能被指定为该酶的抑制剂之一四氢生物蝶呤的结合位点。
