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BIOENERGETICS AND PHOTOSYNTHESIS

Lipoic Acid-Dependent Oxidative Catabolism of -Keto Acids in Mitochondria Provides Evidence for Branched-Chain Amino Acid Catabolism in Arabidopsis¹

Plant Molecular Biology Group, Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia

Lipoic acid-dependent pathways of -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 -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 -keto acids. Pyruvate- and -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 -keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and -keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived -keto acid, -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.

¹ This work was supported by the Australian Research Council Discovery Programme (to A.H.M. and D.A.D.) and by the University of Western Australia (University Postgraduate Scholarship to N.L.T.).

^* Corresponding author; e-mail hmillar{at}cyllene.uwa.edu.au; fax 61–8–9380–1148.

Received November 2, 2003; returned for revision November 21, 2003; accepted December 1, 2003.

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