The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1–4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer.

中文翻译：

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Rubisco 激活酶需要大亚基 N 端的残基来重塑受抑制的​​植物 Rubisco。

光合 CO2 固定酶 1,5-二磷酸核酮糖羧化酶/加氧酶 (Rubisco) 形成死端抑制复合物，同时结合多种糖磷酸盐，包括其底物 1,5-二磷酸核酮糖。Rubisco 可以通过分子伴侣从这种抑制形式中解救出来，该分子伴侣属于与多种细胞活动相关的 ATP 酶（AAA+ 蛋白），称为 Rubisco 激活酶 (Rcas)。在高等植物中发现的绿色型 Rca 的机制已被证明是难以捉摸的，部分原因是直到最近高等植物 Rubiscos 还不能重组表达。确定 Rubisco 和 Rca 之间的相互作用位点对于制定机械假设至关重要。为此，我们在这里纯化并表征了一组 33 种拟南芥 Rubisco 突变体，因为它们具有被 Rca 激活的能力。17 个表面暴露的大亚基残基的突变不会产生在它们与 Rca 的相互作用中受到干扰的变体。相比之下，我们发现 Rca 活动对 Rubisco 大亚基的保守 N 总站中的截断和突变高度敏感。缺少残基 1-4 的大亚基是功能性 Rubiscos，但不能被激活。T5A 和 T7A 取代都导致功能性羧化酶被 Rca 激活较差，表明这些残基的侧链与分子伴侣形成了关键的相互作用。许多其他 AAA+ 蛋白通过将大分子穿过圆盘形六聚体的中心孔来发挥作用。我们的结果与模型一致，在该模型中，Rca 使 Rubisco 大亚基 N 末端瞬时穿过 AAA+ 六聚体的轴向孔。