The acyl–CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p

Abstract

One of the acyl–CoA oxidases from the yeast Yarrowia lipolytica, acyl–CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His) 6 fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 μmol min −1 mg −1 using myristoyl–CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl–CoAs, with a chain length of 10–14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl–CoAs containing more than 14C were also desaturated, if their concentration was below 25 μM or if proteins capable of binding these CoA–esters, such as albumin or β-casein, were added to the assay. These long-chain acyl–CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl–CoA, activity toward hexadecadioyl–CoA, containing a ω-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl–CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl–CoA–esters containing a 2-methyl group, but only the 2S isomer was recognized.