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, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive 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 on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs.
De Bellis
L
L
Dipartimento di Biologia, via Provinciale Lecce-Monteroni, 73100 Lecce, Italy. luigi.debellis@unile.it
Gonzali
S
S
Alpi
A
A
Hayashi
H
H
Hayashi
M
M
Nishimura
M
M
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry*; D042962:Acyl-CoA Oxidase; D017360:Arabidopsis / Q000276:immunology; D029681:Arabidopsis Proteins; D015153:Blotting, Western; D002853:Chromatography, Liquid; D019662:Cucurbitaceae / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D010088:Oxidoreductases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011487:Protein Conformation; D013347:Subcellular Fractions / Q000201:enzymology
D029681:Arabidopsis Proteins; D007527:Isoenzymes; D010088:Oxidoreductases; D044944:Acyl-CoA Dehydrogenases; C467988:ACX3 protein, Arabidopsis; D042962:Acyl-CoA Oxidase; D042964:Acyl-CoA Dehydrogenase