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

Abstract

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 ϩ 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.The enzymatic decarboxylation of ␣-keto organic acids in cells is often facilitated by the cofactor thiamine pyrophosphate (TPP), which acts as a strong nucleophile to attack the carbonyl carbon of ␣-keto acids. The remaining aldehyde can be simply protonated; for example, during acetaldehyde formation by pyruvate decarboxylase (EC 4.1.1.1) or, alternatively, the energy released by the decarboxylation can be coupled to the generation of NADH. The ␣-keto acid decarboxylating dehydrogenase complexes encapsulate this alternative reaction series. These complex structures use E1 (␣-keto acid dehydrogenase), E2 (acyltransferase), and E3 (lipoamide dehydrogenase) enzymes and five cofactors (TPP, CoA, lipoic acid, FAD ϩ , and NAD ϩ ) in their catalytic cycle (Reed, 1974;Mooney et al., 2002). Active-site coupling is used to catalyze decarboxylation of ␣-keto acids (E1 enzyme), esterification of aldehydes to CoA (E2 enzyme), and reduction of NAD ϩ to NADH (E3 enzyme; Reed, 1981;Perham et al., 2002). The most widely studied ␣-keto acid enzyme complexes are the pyruvate dehydrogenase complex (PDC) that regulates the entry of carbon into the tricarboxylic acid (TCA) cycle by the provision of acetyl-CoA and the ␣-ketoglutarate dehydrogenase complex (KGDC) that acts within the TCA cycle to synthesize succinylCoA. B...

Mitochondrial Protein Lipoylation Does Not Exclusively Depend on the mtKAS Pathway of de Novo Fatty Acid Synthesis in Arabidopsis

“…In contrast, we observed distinctly higher lipoylation levels for the PDH and KGDH E2 subunits (about 30% relative to wild type, Fig. 6A), which were identified according to antigenicity and apparent molecular masses (Taylor et al, 2004). These percentages, obtained by scanning software, correspond well with the results from Gly-dependent respiration and enzyme activity measurements discussed above.…”

“…While we cannot entirely exclude the possible presence of a small fraction of HNE-modified peptides in our preparations, limited bias caused by HNE would not weaken but even strengthen our argumentation. H protein of GDC is the dominant lipoylated protein in photosynthesizing plant cells and occurs in large amounts in green leaf mitochondria, representing the major sink for octanoyl chains in these organelles (Wada et al, 1997;Gueguen et al, 2000;Taylor et al, 2004). On the other hand, roots contain only very small amounts of GDC (Douce et al, 2001) and do not require high levels of lipoate biosynthesis.…”

“…First, we have recently shown that a total knockout of GDC is lethal even in nonphotorespiratory conditions, indicating that GDC fulfils a nonreplaceable function in vital metabolic processes other than photorespiration (Engel et al, 2007). Second, in addition to H protein, other mitochondrial proteins of strategic importance, mainly the E2 subunits of the a-ketoacid dehydrogenase complexes PDH and KGDH, also require lipoylation for their activity (Mooney et al, 2002;Taylor et al, 2004). Therefore, the observed features of mtkas mutants indicate significant residual activities of all three multienzyme complexes and point to the possibility that mitochondrial protein lipoylation may not be exclusively dependent on mtKAS.…”

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System

“…Lipoic acid is the cofactor that is essential for pyruvate dehydrogenase (PDH), a-ketoglutarate dehydrogenase (KGDH), branchedchain a-ketoacid dehydrogenase, and the Gly decarboxylase complex (GDC; Taylor et al, 2004). To date, alternative functions for the mtFAS system have not been demonstrated, although its role in detoxifying mitochondrial malonic acid (Guan and Nikolau, 2016) and in remodeling cardiolipins Griebau and Frentzen, 1994) have been suggested.…”

Characterization of the Branched-Chain Amino Acid Aminotransferase Enzyme Family in Tomato

“…Catabolism is believed to be initiated in mitochondria, where the branched-chain keto acid (BCKA) dehydrogenase complex is located (Taylor et al, 2004). The primary fates of BCAAs in plant cells are peptide elongation, glutamate recycling, Glc-and Suc-linked branched-chain esters, branched-chain fatty acid synthesis, and respiration through the synthesis of tricarboxylic acid cycle intermediates (Kandra et al, 1990;Walters and Steffens, 1990;Kroumova et al, 1994;Daschner et al, 1999;Li et al, 2003;Beck et al, 2004;Taylor et al, 2004;Engqvist et al, 2009). BCAA catabolism likely has other functions in plant metabolism.…”