Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis.
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.
Taylor
Nicolas L
NL
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.
Heazlewood
Joshua L
JL
Day
David A
DA
Millar
A Harvey
AH
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000737:chemistry / Q000378:metabolism; D000597:Amino Acids, Branched-Chain / Q000737:chemistry / Q000378:metabolism*; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism*; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D012275:Oryza / Q000201:enzymology; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000502:physiology; D018532:Pisum sativum / Q000201:enzymology; D010940:Plant Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism; D008063:Thioctic Acid / Q000737:chemistry / Q000378:metabolism*; D014633:Valine / Q000737:chemistry / Q000378:metabolism
D000597:Amino Acids, Branched-Chain; D007651:Keto Acids; D009097:Multienzyme Complexes; D010940:Plant Proteins; D011768:Pyruvate Dehydrogenase Complex; D007532:Isoleucine; D008063:Thioctic Acid; D007655:Ketoglutarate Dehydrogenase Complex; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D007930:Leucine; D014633:Valine