Isolation and Characterization of cDNA Clones for the E1β and E2 Subunits of the Branched-chain α-Ketoacid Dehydrogenase Complex in Arabidopsis

Abstract

Branched-chain ␣-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1␤ and E2 subunits of BCKDH, respectively, from Arabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.The mammalian branched-chain ␣-ketoacid dehydrogenase (BCKDH) 1 is a mitochondrial multienzyme complex that is composed of three subunits carrying different enzymatic activities: branched-chain ␣-ketoacid decarboxylase (E1; EC 1.2.4.4), dihydrolipoyl transacylase (E2; no EC number), and dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4). The E1 subunit is further composed of two E1␣ and two E1␤ subunits. This enzyme complex also contains two specific regulatory enzymes, a kinase and a phosphatase (1). E1 catalyzes the oxidative decarboxylation of branched-chain ␣-keto acids, which are derived from branched-chain amino acids by transamination. E2 catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. E3 is a flavoprotein and reoxidizes the reduced lipoyl sulfur residues of the E2 subunit (2).BCKDH in mammals is thought to be a key enzyme in the catabolism of the branched-chain amino acids, i.e. valine, leucine, and isoleucine. As end products, leucine is converted to acetyl-CoA and acetoacetate, valine to succinyl-CoA, and isoleucine to succinyl-CoA and acetyl-CoA (3). Through this pathway, these amino acids serve as substrates for energy production via acetoacetate and succinyl-CoA. It has been firmly established that nutritional conditions play an important role in modulating the activity of BCKDH through a phosphorylation-dephosphorylation mechanism (4). From this point of view, BCKDH is critically important in the pathway for energy utilization, rather than being merely a system for the catabolism of a small group of amino acids.BCKDH has been extensively studied in mammals because defects in the genes for this enzyme cause maple syrup urine disease, an inborn disorder of metabolism in humans (3). In the plant kingdom, however, we have come across only one case reporting the detection...

Identifying Genotype-by-Environment Interactions in the Metabolism of Germinating Arabidopsis Seeds Using Generalized Genetical Genomics

“…This suggests that the degradation pathways provide alternative carbon sources for the plant in extreme conditions. In addition, branched-chain amino acids and their derived a-keto acids are cytotoxic, and preventing accumulation through degradation may be an important detoxification mechanism (Fujiki et al, 2000). Higher levels of both fumarate and malate, as a result of the degradation of a surplus of amino acids, might thus be indicative for larger seed sizes.…”

The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis

“…Two lines of evidence for this role are that the ivd1-2 mutant becomes senescent faster than the wild type in prolonged darkness, and mutants defective in ETFb and ETFQO accumulate more free BCAAs and the IVD substrate isovaleryl-CoA (Ishizaki et al, 2005(Ishizaki et al, , 2006Araújo et al, 2010). In addition, the transcripts of the functionally validated BCAA catabolism genes BCAT2, IVD1, MCCA1, and MCCB1 rapidly increase following transition from light to dark, and this increase is inhibited by Suc (Fujiki et al, 2000;Che et al, 2002;Binder, 2010;Angelovici et al, 2013). These observations suggest that IVD and other BCAA catabolic enzymes contribute to plant fitness under energy-limited conditions.…”

“…Pairs of paralogous genes are annotated as encoding Arabidopsis E1a, E1b, and E3 subunits, and one gene is annotated for E2. These assignments are based on (1) sequence similarity with proteins identified from other organisms, especially mammals, and (2) mitochondrial localization evidence using tandem mass spectrometry, rather than enzyme activities (Fujiki et al, 2000;Mooney et al, 2000;Taylor et al, 2004). Proposed Arabidopsis BCAA catabolism pathway.…”

chy1, an Arabidopsis Mutant with Impaired β-Oxidation, Is Defective in a Peroxisomal β-Hydroxyisobutyryl-CoA Hydrolase

“…However, several potential BCAA catabolic enzymes in Arabidopsis, pea, and soybean have been isolated from mitochondria or contain apparent mitochondrial transit signals (Fig. 4) (6,(53)(54)(55)(56)(57)(58). CHY1 is likely to act in Val catabolism, as it hydrolyzes HIBYL-CoA in vitro (Table II; Fig.…”