Central Research and Development Department, Experimental Station, E.I. du Pont de Nemours & Co., 19898, Wilmington, DE, U.S.A..
Photosynth Res. 1988 Jul;17(1-2):145-57. doi: 10.1007/BF00047686.
The synthesis in Escherichia coli of both the large and small subunits of cereal ribulose bisphosphate carboxylase/oxygenase has been obtained using expression plasmids and bacteriophages. The level and order of synthesis of the large and small subunits were regulated using different promoters, resulting in different subunit pool sizes and ratios that could be controlled in attempts to optimize the conditions for assembly. Neither assembly nor enzyme activity were observed for the higher plant enzyme. In contrast, cyanobacterial large and small subunits can assemble to give an active holoenzyme in Escherichia coli. By the use of deletion plasmids, followed by infection with appropriate phages, it can be demonstrated that the small subunit is essential for catalysis. However, the small subunit is not required for the assembly of a large subunit octomer core in the case of the Synechococcus enzyme; self-assembly of the octomer will occur in an rbcS deletion strain. The cyanobacterial small subunits can be replaced by wheat small subunits to give an active enzyme in Escherichia coli. The hybrid cyanobacterial large/wheat small subunit enzyme has only about 10% of the level of activity of the wild-type enzyme, reflecting the incomplete saturation of the small subunit binding sites on the large subunit octomer, and possibly a mismatch in the subunit interactions of those small subunits that do bind, giving rise to a lower rate of turnover at the active sites.
在大肠杆菌中，通过使用表达质粒和噬菌体，合成了谷物核酮糖二磷酸羧化酶/加氧酶的大亚基和小亚基。大亚基和小亚基的合成水平和顺序受到不同启动子的调控，从而可以控制不同的亚基池大小和比例，以尝试优化组装条件。然而，对于高等植物酶，既没有观察到组装，也没有观察到酶活性。相比之下，蓝细菌的大亚基和小亚基可以在大肠杆菌中组装成具有活性的全酶。通过使用缺失质粒，然后用适当的噬菌体感染，可以证明小亚基对于催化是必需的。然而，在 Synechococcus 酶的情况下，小亚基对于形成具有活性的大亚基八聚体核心的组装并不是必需的；在 rbcS 缺失菌株中，八聚体将自行组装。蓝细菌的小亚基可以被小麦的小亚基取代，在大肠杆菌中形成具有活性的酶。杂交的蓝细菌大亚基/小麦小亚基酶的活性只有野生型酶的约 10%，这反映了大亚基八聚体上的小亚基结合位点不完全饱和，并且可能是那些结合的小亚基的亚基相互作用不匹配，导致活性位点的周转率降低。
