Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component

Abstract

Plastidic acetyl-CoA carboxylase (ACCase; EC 6.4.1.2), which catalyses the synthesis of malonyl-CoA and is the regulatory enzyme of fatty acid synthesis, is activated by light, presumably under redox regulation. To obtain evidence of redox regulation in vivo, the activity of ACCase was examined in pea chloroplasts isolated from plants kept in darkness (dark-ACCase) or after exposure to light for 1 h (light-ACCase) in the presence or absence of a thiol-reducing agent, dithiothreitol (DTT). The protein level was similar for light-ACCase and dark-ACCase, but the activity of light-ACCase in the absence of DTT was approx. 3-fold that of dark-ACCase. The light-ACCase and dark-ACCase were activated approx. 2-fold and 6-fold by DTT respectively, indicating that light-ACCase was in a much more reduced, active form than the dark-ACCase. This is the first demonstration of the light-dependent reduction of ACCase in vivo. Measurement of the activities of ACCase, carboxyltransferase and biotin carboxylase in the presence and absence of DTT, and the thiol-oxidizing agent, 5, 5'-dithiobis-(2-nitrobenzoic) acid, revealed that the carboxyltransferase reaction, but not the biotin carboxylase reaction, was redox-regulated. The cysteine residue(s) responsible for redox regulation probably reside on the carboxyltransferase component. Measurement of the pH dependence of biotin carboxylase and carboxyltransferase activities in the ACCase suggested that both components affect the activity of ACCase in vivo at a physiological pH range. These results suggest that the activation of ACCase by light is caused partly by the pH-dependent activation of two components and by the reductive activation of carboxyltransferase.

Linking Gene Expression and Membrane Lipid Composition ofArabidopsis

“…Whereas FA synthesis is known to be light regulated at the level of ACCase by the redox mechanism (Kozaki and Sasaki, 1999), here we observe that genes involved in FA synthesis are also coordinately regulated at the transcriptional level. This involves the dark-induced downregulation of almost all key pathway enzymes, including elements of the FA synthase complex: KASI, KASIII, hydroxyl-ACP dehydrase, and enoyl-ACP reductase ENR1; the biotin carboxylase subunit of ACCase, malonyl-CoA:ACT malonyltransferase, the longchain acyl-CoA synthetase LACS9, the acyl-ACP synthetase AAE15; and components of the pyruvate dehydrogenase complex: one of the genes for b-PDH (At1g30120) and LPD2 ( Figure 4B).…”

Coordinate Changes in Carbon Partitioning and Plastidial Metabolism during the Development of Oilseed Rape Embryos

“…The regulatory properties of ACCase are increasingly evident (Post-Beittenmiller et al, 1992;Hunter and Ohlrogge, 1998;Kozaki and Sasaki, 1999;Savage and Ohlrogge, 1999), and the role of this enzyme in determining oil content has been demonstrated by increasing the plastidial ACCase activity in embryos of transgenic rapeseed to gain an increase in oil content under certain growth conditions (Roesler et al, 1997). Recently, Bao and Ohlrogge (1999) have shown that the rate of oil synthesis by embryos of oilseed species can be stimulated in vitro by the supply of free fatty acids, providing evidence that the supply of de novo-synthesized fatty acyl groups to the lipid biosynthetic pathway can be a limiting step.…”

Plant Acetyl-CoA Carboxylase: Structure, Biosynthesis, Regulation, and Gene Manipulation for Plant Breeding

“…Addition of the reducing agent dithiothreitol to ACCase showed that CT, but not BC, is activated. 64) Pea recombinant CT expressed in E. coli has properties similar to those of authentic CT and is redox-regulated. 21) Site-directed mutagenesis showed that one cysteine residue in the -CT and one cysteine in the -CT involved in redox regulation form an intermolecular disulfide bond between and subunits.…”