Witter K, Cahill D J, Werner T, Ziegler I, Rödl W, Bacher A, Gütlich M
GSF-Institut für Klinische Molekularbiologie, München, Germany.
Biochem J. 1996 Oct 1;319 ( Pt 1)(Pt 1):27-32. doi: 10.1042/bj3190027.
The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM.
盘基网柄菌的鸟苷三磷酸环化水解酶I（GTP-CH）基因已被克隆并测序。该基因855bp的cDNA包含一个开放阅读框（ORF），编码232个氨基酸，预测分子量约为26kDa。Southern印迹分析表明，盘基网柄菌中存在一个GTP-CH单基因。从染色体DNA对ORF进行PCR扩增并测序表明，盘基网柄菌的GTP-CH基因中存在一个101bp的内含子。该氨基酸序列与人类和酵母酶的氨基酸序列分别具有47%和49%的位置同一性。物种间的大部分序列变异位于蛋白质的N端部分。与大肠杆菌蛋白质的整体同一性明显较低。该酶在大肠杆菌中表达，并作为与大肠杆菌麦芽糖结合蛋白的68kDa融合蛋白进行纯化。盘基网柄菌的GTP-CH热稳定，在60℃时表现出最大活性。GTP的Km值为50μM。
