Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase

Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis. This enz yme in plants is localized in plastids and is believed to be composed of bi otin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been pu rified yet. Accumulated evidence shows that pea plastidic acetyl-CoA carbox ylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a red ox cascade. To understand the reductive activation of carboxyltransferase a t the molecular level here, we obtained the active enzyme composed of decah istidine-tagged (His tag) alpha and beta polypeptides through the expressio n of the pea plastidic carboxyltransferase gene in Escherichia coli. Gel fi ltration showed that the molecular size of the recombinant carboxyltransfer ase is in agreement with that of partially purified carboxyltransferase fro m pea chloroplasts, The catalytic activity of the recombinant enzyme was si milar to that of native carboxyltransferase. These results indicate that th e molecular structure and conformation of recombinant carboxyltransferase r esemble those of its native counterpart and that native carboxyltransferase is indeed composed of alpha and beta polypeptides. This recombinant enzyme was activated by dithiothreitol, a known reductant of S-S bonds, with a pr ofile similar to that of its native counterpart. The recombinant enzyme was activated by reduced thioredoxin-f, a signal transducer of redox potential in chloroplasts under irradiation. Thus, this enzyme was redox-regulated, like that of the native carboxyltransferase.