Evidence for a peroxisomal fatty acid β‐oxidation involving d‐3‐hydroxyacyl‐CoAs

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Abstract
A novel d‐(–)‐3‐hydroxyacyl‐CoA hydro‐lyase, forming 2‐trans‐enoyl‐CoA and formerly designated as epimerase (EC 5.1.2.3), was extracted from fat‐degarding cotyledons of cucumber seedling. The enzyme, called d‐3‐hydroxyacyl‐CoA hydro‐lyase or d‐specific 2‐trans‐enoyl‐CoA hydratase, is shown to be required for the degradation of unsaturated fatty acids that contain double bonds extending from even‐numbered C atoms. The d‐3‐hydroxyacyl‐CoA hydro‐lyase was exlusively localized within peroxisomes. A 10000‐fold purification by chromatography on a hydrophobic matrix, a cation exchanger, on hydroxyapatite and Mono S led to two proteins of apparent homogeneity, both exhibiting Mr of 65000. The d‐3‐hydroxyacyl‐CoA hydro‐lyases are homodimers with slightly differing isoelectric points around pH = 9.0. They catalyze the conversion of 2‐trans‐enoyl‐CoA into d‐3‐hydroxyacyl‐CoA. The reverse reaction was observed but no reaction with 2‐cis‐enoyl‐CoAs or l‐3‐hydroxyacyl‐CoAs. 2‐trans‐Decenoyl‐CoA was converted 10‐times faster than 2‐trans‐butenoyl‐CoA. The conversion of 4‐cis‐decenoyl‐CoA into octenoyl‐CoA was demonstrated in vitro with purified proteins with an assay mixture containing acyl‐CoA oxidase, multifunctional protein, thiolase and the d‐3‐hydroxyacyl‐CoA hydrolyase. Comparisons of enzyme activities present in the cotyledons or isolated peroxisomes clearly show that the pathway via dienoyl‐CoA reductase in much less effective than the sequence involving d‐3‐hydroxyacyl‐CoA hydro‐lyase.

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