Comparative in vitro analyses of recombinant maize starch synthases SSI, SSIIa, and SSIII reveal direct regulatory interactions and thermosensitivity
Comparative in vitro analyses of recombinant maize starch synthases SSI, SSIIa, and SSIII reveal direct regulatory interactions and thermosensitivity
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DOI：
10.1016/j.abb.2016.02.032
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发表时间：
2016-04-15
影响因子：
3.9
通讯作者：
Myers, Alan M.
中科院分区：
文献类型：
--
作者：
Huang, Binquan;Keeling, Peter L.;Myers, Alan M.
淀粉合酶SSI、SSII和SSIII在组装淀粉的支链淀粉成分中发挥作用，但它们的具体作用和协调方式尚不完全清楚。遗传分析表明 SS 类别之间的调节相互作用以及它们之间的物理相互作用是已知的。谷物 SSIII 的 N 末端延伸包含催化结构域之外的多达 1200 个残基，至少部分负责这些相互作用。测试了重组玉米 SSI、SSIIa 和全长或截短的 SSIII 与酶活性相关的功能相互作用。与全长酶相比，氨基末端截短的 SSIII 表现出较低的活性，并且在截短的蛋白质中添加 N 末端可刺激催化活性。 SSIII 和 SSI 显示出负相互作用，降低了重构系统中的总活性。这些数据表明 SSIII 既是催化因素又是调节因素。在 45 摄氏度下短暂孵育后，SSIII 活性降低了约 50%，这表明其在高温生长过程中减少了淀粉积累。测试缓冲效应是为了解决当前有关 SS 机制的争论。无论缓冲系统如何，葡聚糖都会刺激 SSIIa 和 SSIII 反应速率，支持将葡萄糖基单元添加到外源引物底物中的公认机制。 (C) 2016 Elsevier Inc. 保留所有权利。
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 degrees 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. (C) 2016 Elsevier Inc. All rights reserved.