Purification and characterization of a novel pumpkin short-chain acyl-coenzyme A oxidase with structural similarity to acyl-coenzyme A dehydrogenases

A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapati te, affinity, and anion-exchange chromatography. The purified enzyme is a t etrameric protein, consisting of apparently identical 47-kD subunits. The p rotein structure of this oxidase differs from other plant and mammalian ACO Xs, but is similar to the protein structure of mammalian mitochondrial acyl -CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial A CDH. Subcellular organelle separation by sucrose density gradient centrifug ation revealed that the enzyme is localized in glyoxysomes, whereas no immu noreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity o n C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibite d by high concentrations of substrate and it is not inhibited by Triton X-1 00 at concentrations up 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs.