Wang Xiaoqin, Lei Guofeng, Wu Xiaoyu, Wang Fei, Lai Chongde, Li Zhimin
College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, Jiangxi, 330045, China.
College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, Jiangxi, 330045, China; Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, Nanchang, Jiangxi, 330045, China.
Protein Expr Purif. 2017 Nov;139:21-28. doi: 10.1016/j.pep.2017.07.013. Epub 2017 Jul 19.
The sll1981 protein from cyanobacterium Synechocystis sp. PCC6803 had been reported to exhibit acetolactate synthase (ALS) and L-myo-inositol-1-phosphate synthase (MIPS) activities previously. Based on amino acids sequences alignment, sll1981 protein was postulated to function as α-ketoglutarate decarboxylase (α-KGD), which played important role in completing cyanobacterial tricarboxylic acid (TCA) cycle. However the detailed enzymatic kinetics of sll1981 as ALS, MIPS and α-KGD were not determined yet. In this study, the recombinant sll1981 protein was purified from supernatant of E. coli cell and the substrate specificity of sll1981 towards pyruvate, d-glucose-6-phosphate and α-ketoglutarate was examined using homogenous recombinant sll1981. Steady-state kinetics results showed that sll1981 was a dual functional enzyme, which displayed much higher activity as α-KGD than as ALS. At the same time the MIPS activity of sll1981 was not detectable, although it was reported to be as MIPS previously. These findings not only confirmed the previous statement of the function of sll1981 as ALS and disputed the claimed function of sll1981 as MIPS, but also affirmed the new function of sll1981 as α-KGD. Therefore sll1981 was probably a key enzyme in completing the TCA cycle of Synechocystis sp. PCC6803.
先前有报道称，来自集胞藻6803（Synechocystis sp. PCC6803）的sll1981蛋白具有乙酰乳酸合酶（ALS）和L-肌醇-1-磷酸合酶（MIPS）活性。基于氨基酸序列比对，推测sll1981蛋白可作为α-酮戊二酸脱羧酶（α-KGD）发挥作用，该酶在蓝藻三羧酸（TCA）循环中起着重要作用。然而，sll1981作为ALS、MIPS和α-KGD的详细酶动力学尚未确定。在本研究中，从大肠杆菌细胞的上清液中纯化了重组sll1981蛋白，并使用均一的重组sll1981检测了sll1981对丙酮酸、d-葡萄糖-6-磷酸和α-酮戊二酸的底物特异性。稳态动力学结果表明，sll1981是一种双功能酶，其作为α-KGD的活性远高于作为ALS的活性。同时，尽管先前报道sll1981具有MIPS活性，但本研究未检测到其MIPS活性。这些发现不仅证实了先前关于sll1981作为ALS功能的说法，对其作为MIPS的声称功能提出了质疑，还肯定了sll1981作为α-KGD的新功能。因此，sll1981可能是集胞藻6803完成TCA循环的关键酶。
