Expression, purification and characterization of sll1981 protein from cyanobacterium Synechocystis sp. PCC6803

- A method to express recombinant sll1981 in soluble form was established for the first time.
- Recombinant sll1981 was purified to be homogenous with purity of >95%.
- The kinetics of sll1981 as ALS and α-KGD were characterized for the first time.

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.