Properties of hybrid enzymes between Synechococcus large subunits and higher plant small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli.
To explore the function of small subunits of Rubisco, three hybrid enzymes were synthesized in Escherichia coli by construction of a transcriptionally coupled expression system in which the synthetic small subunit gene of rice, tobacco, and wheat, respectively, was cloned downstream from the large subunit gene of Synechococcus sp. PCC6301. These coexpression products were detected by utilizing SDS-PAGE and confirmed by immunoblotting. The amount of carboxylase activity from the intact cells revealed that each higher plant small subunit was able to assemble with the Synechococcus large subunit octamer core to form an active heterologous enzyme in E. coli. However, in these heterologous enzymes, the interaction between large subunits and small subunits was very weak, the small subunit readily dissociated from the large subunit octamer core. A detailed kinetic assay was carried out with the partially purified hybrid enzymes. Compared to Synechococcus Rubisco, the activity of rice, tobacco, and wheat hybrid Rubisco decreased to 37, 61, and 37% of the original activity, respectively. These hybrid enzymes showed a greater affinity for CO2 and RuBP than Synechococcus Rubisco. The specificity factor of the three hybrid Rubiscos was 98, 84, and 76%, respectively, of the original. These results indicate for the first time that the small subunit contributes to the stability, catalytic efficiency, and CO2/O2 specificity of Rubisco together, which suggests that small subunits may be fruitful targets for engineering an improved Rubisco. Meanwhile, we found that sorbitol in the culture of induced cells promoted the production of active assembled enzyme and shortened the time to reach maximal expression.
Wang
Y L
YL
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Shanghai, 200032, China.
Zhou
J H
JH
Wang
Y F
YF
Bao
J S
JS
Chen
H B
HB
D000458:Cyanobacteria / Q000201:enzymology*; D002523:Edible Grain / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D010957:Plasmids; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D014026:Nicotiana / Q000201:enzymology*
D021122:Protein Subunits; D011994:Recombinant Proteins; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase