The Mitochondrial Isovaleryl-Coenzyme A Dehydrogenase of Arabidopsis Oxidizes Intermediates of Leucine and Valine Catabolism

Abstract

We recently identified a cDNA encoding a putative isovaleryl-coenzyme A (CoA) dehydrogenase in Arabidopsis (AtIVD). In animals, this homotetrameric enzyme is located in mitochondria and catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA as an intermediate step in the leucine (Leu) catabolic pathway. Expression of AtIVD:smGFP4 fusion proteins in tobacco (Nicotiana tabacum) protoplasts and biochemical studies now demonstrate the in vivo import of the plant isovaleryl-CoA dehydrogenase (IVD) into mitochondria and the enzyme in the matrix of these organelles. Two-dimensional separation of mitochondrial proteins by blue native and SDS-PAGE and size determination of the native and overexpressed proteins suggest homodimers to be the dominant form of the plant IVD. Northern-blot hybridization and studies in transgenic Arabidopsis plants expressing Ativd promoter:gus constructs reveal strong expression of this gene in seedlings and young plants grown in the absence of sucrose, whereas promoter activity in almost all tissues is strongly inhibited by exogeneously added sucrose. Substrate specificity tests with AtIVD expressed in Escherichia coli indicate a strong preference toward isovaleryl-CoA but surprisingly also show considerable activity with isobutyryl-CoA. This strongly indicates a commitment of the enzyme in Leu catabolism, but the activity observed with isobutyryl-CoA also suggests a parallel involvement of the enzyme in the dehydrogenation of intermediates of the valine degradation pathway. Such a dual activity has not been observed with the animal IVD and may suggest a novel connection of the Leu and valine catabolism in plants.

The Critical Role of Arabidopsis Electron-Transfer Flavoprotein:Ubiquinone Oxidoreductase during Dark-Induced Starvation

“…In Arabidopsis, isovaleryl-CoA dehydrogenase is localized in plant mitochondria (Daschner et al, 2001;Taylor et al, 2004), and our metabolite profiling data ( Figure 5) demonstrated a marked accumulation of the isovaleryl-CoA dehydrogenase substrate, isovaleryl-CoA, in etfqo mutants, suggesting dysfunction of isovaleryl-CoA dehydrogenase in these mutants. These results further support the existence of the electron transfer cascade from isovaleryl-CoA dehydrogenase to ETF, ETF to ETFQO, and ETFQO to ubiquinone in the electron transport chain of plant mitochondria as reported in mammals , although the direct interaction of these four components has yet to be confirmed.…”

“…Such a hypothesis is consistent with the observations that the peroxisomal short-chain acyl-CoA oxidase is able to oxidize isobutyryl-CoA (Hayashi et al, 1999), and the demonstration that several other enzymes involved in Ile and Val catabolism are localized in peroxisomes in Arabidopsis (Lange et al, 2004;Taylor et al, 2004). The mitochondrial isovaleryl-CoA dehydrogenase does utilize 2-methyl-butyryl-CoA as a substrate in vitro; however, the K m value of the enzyme for the isobutyryl-CoA is ;10 times larger than that for isovaleryl-CoA (Daschner et al, 2001). Since the relative concentration of Val is only half that of Leu, the large difference in K m values suggests that isovalerylCoA dehydrogenase is not involved in Val oxidation in vivo.…”

“…During dark adaptation, the transcripts from a light-regulated photosynthetic gene, Cab4, gradually declined ( Figure 2) (Fujiki et al, 2001), whereas the ETFQO transcripts accumulated to very high levels ( Figure 2). As a positive control, the transcript levels of the isovaleryl-CoA dehydrogenase gene (IVD) and the E1a subunit of branched-chain a-keto dehydrogenase gene (BCKDH), which have previously been shown to be induced by sucrose starvation (Daschner et al, 2001;Fujiki et al, 2001), were analyzed. The IVD and BCKDH transcripts also accumulated in the dark-adapted leaves (Figure 2), suggesting that expression of the ETFQO gene is also induced by sucrose starvation following prolonged darkness.…”

BRANCHED-CHAIN AMINOTRANSFERASE4 Is Part of the Chain Elongation Pathway in the Biosynthesis of Methionine-Derived Glucosinolates inArabidopsis

“…The insert was removed from this plasmid by digestion with XbaI and SmaI and cloned in frame upstream of the GFP gene in the vector psmGFP4 (Davis and Vierstra, 1998). Transformation of tobacco (Nicotiana tabacum) protoplasts and fluorescence microscopy were done as described before (Koop et al, 1996;Dä schner et al, 2001;Diebold et al, 2002). All standard methods used were performed according to established protocols (Sambrook et al, 1989).…”

Gene expression in plant mitochondria: transcriptional and post–transcriptional control

“…This polypeptide stimulates in vitro transcriptional activity from a wheat cox2 mitochondrial promoter and is thus a very promising candidate for a bona fide promoterbinding protein. In pea (Pisum sativum) a 43 kDa protein was purified from mitochondria by its promoter-binding properties, which shows high similarity to isovaleryl CoA dehydrogenase enzymes from other organisms (Däschner et al 2001 could be an interesting example of a dual-function protein, which could connect metabolic activities and requirements with gene expression in mitochondria. Such bifunctional molecular connections have been identified in yeast mitochondria, where several proteins are involved in gene expression, as well as in metabolic and energytransforming processes.…”