Huang Binquan, Keeling Peter L, Hennen-Bierwagen Tracie A, Myers Alan M
Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Center for Biorenewable Chemicals, 1140K Biorenewables Research Laboratory, Iowa State University, Ames, IA 50011, USA.
Arch Biochem Biophys. 2016 Apr 15;596:63-72. doi: 10.1016/j.abb.2016.02.032. Epub 2016 Mar 3.
Starch synthases SSI, SSII, and SSIII function in assembling the amylopectin component of starch, but their specific roles and means of coordination are not fully understood. Genetic analyses indicate regulatory interactions among SS classes, and physical interactions among them are known. The N terminal extension of cereal SSIII, comprising up to 1200 residues beyond the catalytic domain, is responsible at least in part for these interactions. Recombinant maize SSI, SSIIa, and full-length or truncated SSIII, were tested for functional interactions regarding enzymatic activity. Amino-terminal truncated SSIII exhibited reduced activity compared to full-length enzyme, and addition of the N terminus to the truncated protein stimulated catalytic activity. SSIII and SSI displayed a negative interaction that reduced total activity in a reconstituted system. These data demonstrate that SSIII is both a catalytic and regulatory factor. SSIII activity was reduced by approximately 50% after brief incubation at 45 °C, suggesting a role in reduced starch accumulation during growth in high temperatures. Buffer effects were tested to address a current debate regarding the SS mechanism. Glucan stimulated the SSIIa and SSIII reaction rate regardless of the buffer system, supporting the accepted mechanism in which glucosyl units are added to exogenous primer substrates.
淀粉合成酶SSI、SSII和SSIII在组装淀粉的支链淀粉成分中发挥作用，但其具体作用和协调方式尚未完全明确。遗传分析表明不同类别的淀粉合成酶之间存在调控相互作用，且已知它们之间存在物理相互作用。谷物SSIII的N端延伸区，在催化结构域之外包含多达1200个残基，至少部分负责这些相互作用。对重组玉米SSI、SSIIa以及全长或截短的SSIII进行了酶活性方面的功能相互作用测试。与全长酶相比，氨基端截短的SSIII活性降低，而将N端添加到截短蛋白上可刺激催化活性。SSIII和SSI表现出负相互作用，在重构系统中降低了总活性。这些数据表明SSIII既是一种催化因子也是一种调控因子。在45°C短暂孵育后，SSIII活性降低了约50%，这表明其在高温生长期间淀粉积累减少中发挥作用。测试了缓冲液效应以解决当前关于淀粉合成酶机制的争论。无论缓冲液系统如何，葡聚糖均刺激了SSIIa和SSIII的反应速率，支持了将葡萄糖基单元添加到外源引物底物上的公认机制。
