Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase.
Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis. This enzyme in plants is localized in plastids and is believed to be composed of biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been purified yet. Accumulated evidence shows that pea plastidic acetyl-CoA carboxylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a redox cascade. To understand the reductive activation of carboxyltransferase at the molecular level here, we obtained the active enzyme composed of decahistidine-tagged (His tag) alpha and beta polypeptides through the expression of the pea plastidic carboxyltransferase gene in Escherichia coli. Gel filtration showed that the molecular size of the recombinant carboxyltransferase is in agreement with that of partially purified carboxyltransferase from pea chloroplasts. The catalytic activity of the recombinant enzyme was similar to that of native carboxyltransferase. These results indicate that the molecular structure and conformation of recombinant carboxyltransferase resemble 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 profile 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.
Kozaki
A
A
Laboratory of Plant Molecular Biology, Graduate School of Agricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Kamada
K
K
Nagano
Y
Y
Iguchi
H
H
Sasaki
Y
Y
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000494:pharmacology; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D018087:Plastids / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D002352:Carrier Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein; D004229:Dithiothreitol