Rashid Naeem, Cornista Joel, Ezaki Satoshi, Fukui Toshiaki, Atomi Haruyuki, Imanaka Tadayuki
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
J Bacteriol. 2002 Feb;184(3):777-84. doi: 10.1128/JB.184.3.777-784.2002.
A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.
从嗜热栖热菌KOD1（CGT(Tk)）中鉴定并表征了一种编码环糊精葡糖基转移酶（CGTase）的基因。该基因（cgt(Tk)）编码一个含有713个氨基酸残基的蛋白质，该蛋白质具有在α-淀粉酶家族所有成员中都存在的四个保守区域。然而，对应于先前已知CGTases的结构域E的C末端结构域显示出完全不同的一级结构。为了阐明该基因产物的催化功能，通过阴离子交换色谱法纯化了重组酶，并研究了其酶学性质。该酶表现出显著的淀粉降解活性（750 U/mg蛋白质），最佳温度和pH分别为80℃和5.5至6.0。Ca(2+)的存在增强了酶活性，并将最佳温度提高到85至90℃。添加Ca(2+)后，该酶表现出极高的热稳定性，在85℃下80分钟后酶活性几乎没有损失，在100℃下的半衰期为20分钟。CGT(Tk)可以水解可溶性淀粉和糖原，但不能水解支链淀粉。最重要的是，尽管CGT(Tk)具有独特的C末端结构域，但我们发现该蛋白质也表现出显著的CGTase活性，主要产物为β-环糊精。为了确定C末端区域是否参与CGTase活性，我们分析了一个缺失23个C末端氨基酸残基的截短蛋白（CGT(Tk)DeltaC）。CGT(Tk)DeltaC在淀粉结合活性、底物特异性和热稳定性方面表现出相似的性质，但出乎意料地显示出比亲本CGT(Tk)更高的淀粉降解活性。相反，CGT(Tk)DeltaC的环化活性被消除。结果表明，结构新颖的C末端结构域的存在对于CGT(Tk)正确催化环化反应至关重要。
