Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in Plants*

Kerry A. Rouhier, Kenyon College


The subcellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using13C NMR, accumulation of β-hydroxypropionate from [2-13C]propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants, including both monocots and dicots. Examination of coding sequences and subcellular targeting elements indicated that the completed genome of A. thalianalikely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, Arabidopsis mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to β-hydroxypropionate by a modified β-oxidation pathway. The chy1–3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of β-hydroxyisobutyrate from exogenous isobutyrate, but not the accumulation of β-hydroxypropionate from exogenous propionate. The chy1–3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine. 13C NMR analysis of Arabidopsis seedlings exposed to [U-13C]valine did not show an accumulation of β-hydroxypropionate. No evidence was observed for a modified β-oxidation of valine. 13C NMR analysis showed that valine was converted to leucine through the production of α-ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified β-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine. Previous SectionNext Section

Propionate, in the form of propionyl-CoA, is produced from a number of metabolic precursors in higher eukaryotes. It is the final product of odd-chain fatty acid β-oxidation (1). It is also produced during the catabolism of several amino acids, including isoleucine, methionine, and valine (1, 2). Propionyl-CoA is also a final product of metabolism of the branched acid, phytanic acid, derived from the degradation of chlorophyll (3). Aside from a basic understanding of metabolic biochemistry, the anabolic and catabolic pathways for propionyl-CoA are also of considerable importance in metabolic engineering of polyhydroxyalkanoates in plants, especially in the production of mixed polyhydroxyalkanoate polymers that have relied on the use of propionyl-CoA as a metabolic intermediate (4, 5). Several pathways have been confirmed for the catabolism of propionyl-CoA (68). Bacteria and yeast utilize a 2-methylcitrate pathway with reactions analogous to those of the tricarboxylic acid cycle and glyoxylate cycle (6). Mammals use a well established biotin and B12-dependent pathway for conversion of propionyl-CoA to succinyl-CoA (7, 8).

Although plants have the same capacity described above to produce propionyl-CoA, their catabolic metabolism of propionyl-CoA is not clearly understood, and there are several conflicting reports regarding enzyme activities for different pathways. Examination of plant genomes does not reveal the presence of obvious orthologues corresponding to enzymes from either the bacterial or mammalian pathways. Plant genomes do code for a number of biotin-dependent carboxylases but none with a high level of homology to known propionyl-CoA carboxylases. Nevertheless, propionyl-CoA carboxylase activity has been demonstrated in plant extracts (9), raising some confusion about this enzyme. In the absence of a specific propionyl-CoA carboxylase, this reaction could be catalyzed by either an acetyl-CoA carboxylase or a methylcrotonyl-CoA carboxylase as a side reaction or as a secondary function of such an enzyme. Two separate isoforms of acetyl-CoA carboxylase are present in higher plants (10) as well as methylcrotonyl-CoA carboxylase (9). A problem would then arise as to the subsequent fate of methylmalonyl-CoA derived from carboxylation of propionyl-CoA in the absence of enzymes that could further metabolize methylmalonyl-CoA such as a racemase and a mutase, which appear to be absent in plants. Furthermore, whether B12 is involved in plant metabolism is also not clear. Although plants are generally considered to lack B12 (1113), there are reports in the literature of B12 and other corrin compounds from plant sources (14, 15).

Several other possible pathways have been proposed for the metabolism of propionyl-CoA in various organisms. These include conjugation to glyoxylate to produce either hydroxyglutarate (16) or 3-methylmalate (17), and conversion to acrylyl-CoA and β-hydroxypropionyl-CoA by enzymes similar to those used in a modified β-oxidation pathway for valine catabolism (2). Several studies have reported the production of β-hydroxypropionate from either acrylate or propionate by a modified β-oxidation pathway in either whole plants or isolated peroxisomes (2, 12, 13). Although this provides significant evidence for such a pathway, it is still not clear whether this is the primary pathway or the only functional pathway for metabolism of propionyl-CoA in plants. Furthermore it is not clear whether this modified β-oxidation pathway occurs exclusively in peroxisomes or if it is also in mitochondria. This is an important question as propionyl-CoA can be produced by multiple pathways that are expected to occur in both organelles.

With the completed genome sequence of Arabidopsis thaliana and genome databases of numerous other plant species, it is now possible to consider not only the pathways for catabolism but also the genes involved and the subcellular localization of the corresponding enzymes. Examination of genes coding for key enzymes of a modified β-oxidation pathway for propionyl-CoA suggests that the catabolic disposal of propionyl-CoA may be exclusively peroxisomal in plants and that β-hydroxypropionate may be the final product of this metabolism. This concept is consistent with previous reports of β-hydroxypropionate production in plants (2, 13).

In the present study, we have confirmed the accumulation of β-hydroxypropionate in A. thaliana and other plant species using [2-13C]propionate by 13C NMR spectroscopy. The peroxisomal production of β-hydroxyisobutyrate from exogenous isobutyrate was also examined. Plant genomes code for both mitochondrial and peroxisomal forms of β-hydroxyacyl-CoA hydrolase, a key enzyme in this pathway, which also hydrolyzes branched-chain hydroxyacyl-CoA esters (1821). It has been suggested that these peroxisomal hydrolases may be functionally important for valine catabolism. The present study shows that β-hydroxypropionate is produced from exogenous propionate but not from propionyl-CoA derived from valine and that mutation of a peroxisomal hydroxyacyl-CoA hydrolase results in increased sensitivity to propionate but not to valine. This mutation also abolished the accumulation of β-hydroxyisobutyrate from exogenous isobutyrate. 13C NMR studies of A. thaliana seedlings treated with [U-13C]valine showed that the major pathway for valine metabolism was not through a modified β-oxidation pathway, but through the conversion to leucine.

These data support a pathway in peroxisomes for the β-oxidation of isobutyryl-CoA and propionyl-CoA from metabolic sources other than valine. A better understanding of this pathway may be valuable for the manipulation of this metabolism in the engineered synthesis of polyhydroxyalkanoates containing these acids (4, 5).