Lin Shu-Chuan, Liu Wei-Ting, Liu Shi-Hwei, Chou Wei-I, Hsiung Bor-Kai, Lin I-Ping, Sheu Chia-Chin, Dah-Tsyr Chang Margaret
Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
BMC Biochem. 2007 Jun 25;8:9. doi: 10.1186/1471-2091-8-9.
Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132-167) connects the two functional domains, but its structural and functional roles are unclear.
To characterize the linker sequences of RoGA and its involvement in protein expression, a number of RoGA variants containing deletions and mutations were constructed and expressed in Saccharomyces cerevisiae. Deletion analyses demonstrate that the linker region, especially within residues 161 to 167, is required for protein expression. In addition, site-directed mutagenesis and deglycosylation studies reveal that the linker region of RoGA contains both N- and O-linked carbohydrate moieties, and the N-linked oligosaccharides play a major role in the formation of active enzyme. Although the linker segment itself appears to have no ordered secondary structural conformation, the flexible region indeed contributes to the stabilization of functional N- and C-terminal domains.
Our data provide direct evidence that the length, composition, and glycosylation of the interdomain linker play a central role in the structure and function of RoGA.
米根霉葡糖淀粉酶（RoGA）由三个结构域组成：氨基（N）末端的生淀粉结合结构域（SBD）、一个糖基化连接结构域和羧基（C）末端催化结构域。36个氨基酸的连接区域（第132 - 167位氨基酸残基）连接这两个功能结构域，但其结构和功能作用尚不清楚。
为了表征RoGA的连接序列及其在蛋白质表达中的作用，构建了许多包含缺失和突变的RoGA变体，并在酿酒酵母中进行表达。缺失分析表明，连接区域，特别是第161至167位氨基酸残基对于蛋白质表达是必需的。此外，定点诱变和去糖基化研究表明，RoGA的连接区域同时含有N - 连接和O - 连接的碳水化合物部分，并且N - 连接的寡糖在活性酶的形成中起主要作用。虽然连接片段本身似乎没有有序的二级结构构象，但该柔性区域确实有助于功能性N末端和C末端结构域的稳定。
我们的数据提供了直接证据，表明结构域间连接区的长度、组成和糖基化在RoGA的结构和功能中起核心作用。
