Purification, characterization and cloning of isovaleryl‐CoA dehydrogenase from higher plant mitochondria

Abstract

Between the different types of Acyl-CoA dehydrogenases (ACADs), those specific for branched chain acyl-CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl-CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl-CoA dehydrogenases involved in fatty acid b-oxidation. In plants, fatty acid b-oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched-chain amino acids had not been definitely assigned.Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl-CoA comparable to those of the mammalian enzyme. It is not active towards straight-chain acyl-CoA substrates tested, but significant activity was also found with isobutyryl-CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino-acid catabolism in plant development.Keywords: plant mitochondria; isovaleryl-CoA dehydrogenase; leucine catabolism.The desaturation of the C 2 -C 3 bond of acyl-CoA esters to 2-trans-enoyl-CoA is an important step in the b-oxidation of fatty acids [1,2] and in the catabolism of branchedchain amino acids, leucine, isoleucine and valine [3,4]. In eukaryotes, this reaction can be catalyzed by two distinct type of enzymes: mitochondrial acyl-CoA dehydrogenases (ACADs), which feed reducing equivalents to the ubiquinone mitochondrial pool [5], and by peroxisomal acyl-CoA oxidases (ACOXs), which pass electrons directly on to molecular oxygen producing H 2 O 2 [2]. Both types of enzymes utilize FAD as cofactor. In mammalian tissues, b-oxidation takes place in both peroxisomes and mitochondria. In contrast, in higher plants this catabolism takes place mainly in peroxisomes [6±8], and the existence in higher plants of an additional mitochondrial b-oxidation system (and thus of ACAD enzymes) has often been considered controversial [9±11]. However, inhibition of mitochondrial b-oxidation by respiratory-chain inhibitors [9,12] strongly suggested that higher-plant mitochondria possess ACAD activities, which were identified in mitochondria from carbohydrate-starved maize root tips and sunflower embryos [12,13]. Moreover, putative plant ACAD genes could be identified on the basis of their sequence similarity with the corresponding mammalian enzymes [13]. However, a number of these putative ACADs have eventually been characterized as peroxisomal ACOXs [11,14].Among ACAD activities typically found in the mitochondria of animal cells, those specific for branched-chain substrates are involved in the catabolism of amino acids, in which the isobutyryl-CoA, 2-methyl-butyryl-CoA, and isovaleryl-CoA catabolites of Val, Ile, and Leu, respectively, und...

Convergent Evolution of a 2-Methylbutyryl-CoA Dehydrogenase from Isovaleryl-CoA Dehydrogenase in Solanum tuberosum

“…Molecular Mass of the St-IVD1 and St-IVD2 Gene ProductsPartially purified native potato IVD was previously reported to be a dimer of 85-90 kDa as was recombinant Arabidopsis IVD from an E. coli crude cellular extract (12,13). In the present experiments, the apparent molecular masses were estimated to be 188 and 146 kDa for the purified St-IVD1 and St-IVD2 gene products, respectively, using high pressure liquid chromatography gel filtration (Fig.…”

“…IVDs have been partially purified from potato and Arabidopsis and confirmed to have maximal enzyme activity with isovaleryl-CoA (12,13). Potato and Arabidopsis IVDs are unusual in that they were reported to be homodimers, and both appeared to have significant enzyme activity with isobutyryl-CoA, which is not used as substrate by either mammalian or Caenorhabditis elegans IVDs.…”

Transcriptional analysis between two wheat near-isogenic lines contrasting in aluminum tolerance under aluminum stress

“…In addition, more transcripts of a putative IVD were detected in Chisholm-S at 6 h of Al exposure. This enzyme catalyzes degradation of leucine and valine for energy metabolism following carbohydrate depletion, and it can be induced in carbohydratestarved maize root (Faivre-Nitschke et al 2001). These results imply that Al stress may set oV a cascade of degradation reactions of fatty acids, amino acids, and starch in an Al-sensitive genotype.…”

Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in Plants

“…A primary obstacle for this pathway is the apparent lack of a mitochondrial acyl-CoA dehydrogenase functional for oxidation of propionyl-CoA (27). An isovaleryl-CoA dehydrogenase has been characterized from several plant species, including A. thaliana (28,33,34). However, this enzyme has been shown to be highly specific for branched-chain acyl groups and was inactive with short-chain acyl-CoA substrates like butyryl-CoA and propionyl-CoA (28).…”