Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Abstract

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.

Figure 1.

Identification of proteins that react with antilipoic acid antibodies in Arabidopsis, rice, and pea mitochondria. A, Western blot of two-dimensional IEF/SDS-PAGE separation of Arabidopsis mitochondrial proteins probed with antilipoic acid antibodies. B, Two-dimensional IEF/SDS-PAGE separation of Arabidopsis mitochondrial proteins stained with colloidal Coomassie solution. C, Western blot of two-dimensional IEF/SDS-PAGE separation of rice mitochondrial proteins probed with antilipoic acid antibodies. D, Two-dimensional IEF/SDS-PAGE separation of rice mitochondrial proteins stained with colloidal Coomassie solution. E, Western blot of two-dimensional IEF/SDS-PAGE separation of pea mitochondrial proteins probed with antilipoic acid antibodies. F, Two-dimensional IEF/SDS-PAGE separation of pea mitochondrial proteins stained with colloidal Coomassie solution. Numbers on left of A, C, and E represent the apparent molecular masses. Numbers above A to F represent pI of separated protein spots.

Figure 2.

Arabidopsis mitochondrial localized components involved in the catabolism of branched amino acids. Steps in black involve enzymes that have been localized to Arabidopsis mitochondria and include BCAT, BCKDC, E-CoAH, ETF, hydroxymethylglutaryl-CoA lyase (HMG-CoAL), IVD, acetyl-CoA C-acetyltransferase (A-CoAAT), MCCase, and methylmalonate-semialdehyde dehydrogenase (MMSDH). Steps in gray show enzymes that may be localized elsewhere and include 3-hydroxyisobutyryl-CoA hydrolase (HIB-CoAH), 3-hydroxybutyryl dehydrogenase (HIBDH), and 3-hydroxy-2-methylbutyryl-CoA dehydrogenase (HMB-CoADH). Bracketed amino acids indicate divergent pathways specific to those amino acids.