A Novel Acyl-CoA Oxidase That Can Oxidize Short-chain Acyl-CoA in Plant Peroxisomes

Abstract

Short-chain acyl-CoA oxidases are ␤-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete ␤-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform ␤-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C 8 ). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C 4 ), hexanoyl-CoA (C 6 ), and octanoyl-CoA (C 8 ). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acylCoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid ␤-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid ␤-oxidation in plant peroxisomes, and that by the cooperative action of long-and short-chain acylCoA oxidases, plant peroxisomes are capable of performing the complete ␤-oxidation of acyl-CoA.Oilseed plants convert reserve oil to sucrose after germination. This unique type of gluconeogenesis occurs in the storage tissues of oilseeds, such as endosperms or cotyledons (1). The metabolic pathway involves many enzymes in several subcellular compartments, including lipid bodies, glyoxysomes (a specialized peroxisome), mitochondria, and the cytosol. Within the entire gluconeogenic pathway, the conversion of a fatty acid to succinate takes place within the glyoxysomes, which contain enzymes for fatty acid ␤-oxidation and the glyoxylate cycle.Glyoxysomes and leaf peroxisomes are members of a group of organelles called peroxisomes (2). In glyoxysomes, fatty acids are first activated to fatty acyl-CoA by fatty acyl-CoA synthetase (3). Fatty acyl-CoA is the substrate for fatty acid ␤-oxidation, which consists of four enzymatic reactions (4). The first reaction is catalyzed by acyl-CoA oxidase. The second and third enzymatic reactions are catalyzed by a single enzyme that possesses enoyl-CoA hydratase and ␤-hydroxyacyl-CoA dehydrogenase activities (5). The fourth reaction is catalyzed by 3-ketoacyl-CoA thiolase (referred to as thiolase below) (6). Acetyl-CoA, an end product of fatty acid ␤-oxidation, is metabolized further to produce succinate by the glyoxylate cycle.In mammalian cells, both peroxisomes and mitochondria contain a functional fatty acid ␤-oxidation system. In peroxisomes, the first enzyme of fatty acid ␤-oxidation, acyl-CoA oxidase, donates...

Molecular Characterization of an Arabidopsis Acyl-Coenzyme A Synthetase Localized on Glyoxysomal Membranes

“…5). Similar patterns have been observed previously for other plant glyoxysomal enzymes such as malate synthase (Mori and Nishimura, 1989), citrate synthase (Kato et al, 1995), and SACX (Hayashi et al, 1999).…”

“…We have previously studied enzymes of ␤-oxidation and the glyoxylate cycle in pumpkin (Cucurbita sp. Kurokawa Amakuri) and Arabidopsis in detail (Mori and Nishimura, 1989;Kato et al, 1996bKato et al, , 1998Hayashi et al, 1998aHayashi et al, , 1998bHayashi et al, , 1999De Bellis et al, 1999Fukao et al, 2002). We extended our study to plant glyoxysomal ACSs.…”

Gene-Specific Involvement of β-Oxidation in Wound-Activated Responses in Arabidopsis

“…The Arabidopis genome (Arabidopsis Genome Initiative [AGI], 2000), includes six genes encoding ACXs, ACX1 and ACX2 (Hooks et al, 1999); ACX3 (Eastmond et al, 2000;Froman et al, 2000); ACX4 (Hayashi et al, 1999); and the still uncharacterized homologs of ACX1 and ACX3, which we have named ACX1.2, and ACX3.2 (ACX5 and ACX6, respectively, in Rylott et al, 2003); at least two MFPs, AIM1 and MFP2 (Richmond and Bleecker, 1999); and three KATs, PED1/KAT2 (Hayashi et al, 1998;Germain et al, 2001), PKT2/KAT5 (Germain et al, 2001), and KAT1, a homolog of PED1/KAT2 in chromosome 1. Although b-oxidation has been traditionally considered as a catabolic machinery devoted to the production of energy through fatty acid degradation, it can be also considered as a processing system to convert complex precursors into simpler molecules.…”

Expression, purification and crystallization of two peroxisomal acyl-CoA oxidases fromArabidopsis thaliana

“…ACX4 is a short-chainspeci®c ACX that shows maximal activity on C 6 -CoA (Hayashi et al, 2002). Phylogenetic studies show that ACX4 is more closely related to the mitochondrial ACADs than to other ACXs and, like the ACADs, ACX4 has been characterized as a homotetrameric enzyme (Hayashi et al, 1999).…”

“…Within this ACX family, each isozyme seems to be devoted to the oxidation of acyl-CoAs of speci®c chain lengths (Kirsch et al, 1987). So far, four different ACXs have been identi®ed in the model oil-seed plant Arabidopsis thaliana (Froman et al, 2000;Hayashi et al, 1999;Hooks et al, 1999).…”