Microbial Chemistry, Department of Chemistry - Ångström, Uppsala University, P.O. Box 523, SE-751 20, Uppsala, Sweden.
Department of Cell and Molecular Biology, BMC, Uppsala University, P.O. Box 596, SE-751 24, Uppsala, Sweden.
Sci Rep. 2020 Feb 27;10(1):3607. doi: 10.1038/s41598-020-60249-2.
Phosphoenolpyruvate carboxylase (PEPc) is an essential enzyme in plants. A photosynthetic form is present both as dimer and tetramer in C4 and CAM metabolism. Additionally, non-photosynthetic PEPcs are also present. The single, non-photosynthetic PEPc of the unicellular cyanobacterium Synechococcus PCC 7002 (Synechococcus), involved in the TCA cycle, was examined. Using size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS), we observed that PEPc in Synechococcus exists as both a dimer and a tetramer. This is the first demonstration of two different oligomerization states of a non-photosynthetic PEPc. High concentration of Mg, the substrate PEP and a combination of low concentration of Mg and HCO induced the tetramer form of the carboxylase. Using SEC-SAXS analysis, we showed that the oligomerization state of the carboxylase is concentration dependent and that, among the available crystal structures of PEPc, the scattering profile of PEPc of Synechococcus agrees best with the structure of PEPc from Escherichia coli. In addition, the kinetics of the tetramer purified in presence of Mg using SEC, and of the mixed population purified in presence of Mg using a Strep-tagged column were examined. Moreover, the enzyme showed interesting allosteric regulation, being activated by succinate and inhibited by glutamine, and not affected by either malate, 2-oxoglutarate, aspartic acid or citric acid.
磷酸烯醇式丙酮酸羧化酶（PEPc）是植物中一种必需的酶。在 C4 和 CAM 代谢中，存在着二聚体和四聚体两种光合形式的 PEPc。此外，还存在非光合的 PEPc。本文研究了参与三羧酸循环的单细胞蓝藻集胞藻 PCC 7002（Synechococcus）中的单一非光合 PEPc。通过大小排阻色谱（SEC）和小角 X 射线散射（SAXS），我们观察到 PEPc 在 Synechococcus 中既存在二聚体形式，也存在四聚体形式。这是首次证明非光合 PEPc 存在两种不同的寡聚化状态。高浓度的 Mg、底物 PEP，以及低浓度的 Mg 和 HCO 的组合，诱导了羧化酶的四聚体形式。通过 SEC-SAXS 分析，我们表明羧化酶的寡聚化状态与浓度有关，在现有的 PEPc 晶体结构中，Synechococcus PEPc 的散射谱与大肠杆菌 PEPc 的结构最为吻合。此外，我们还通过 SEC 研究了在 Mg 存在下纯化的四聚体的动力学，以及通过 Strep 标记柱在 Mg 存在下纯化的混合种群的动力学。此外，该酶表现出有趣的变构调节，被琥珀酸激活，被谷氨酰胺抑制，不受苹果酸、2-酮戊二酸、天冬氨酸或柠檬酸的影响。
