Evidence for the mitochondrial biosynthesis of 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid in the yeast Dipodascopsis uninucleata

The biosynthesis of 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE ) from arachidonic acid (20:4n-6) by the hyphal-forming yeast, Dipodascopsi s uninucleata, in cell-free enzyme extracts required CoASH, ATP, NAD(+) and Mg2+; 3R-HETE was present as the CoA derivative in enzyme extracts and its biosynthesis was associated with mitochondria. Its synthesis was high from arachidonoyl-CoA (15% conversion of the substrate; 22 nmol mg protein(-1). h), but significantly higher from trans-2-arachidonoyl-CoA (53 nmol mg prot ein(-1).min). Aspirin, an inhibitor of prostaglandin endoperoxide synthase synthase (cyclooxygenase), did not significantly inhibit 3R-HETE biosynthes is in enzyme extracts, as opposed to antimycin A (46% inhibition). The chir ality of 3-HETE was 95% R and 5% S. 3R-HETE has the same chirality as the p roducts of peroxisomal enoyl-CoA hydratases of Neurospora crassa and Saccha romyces cerevisiae; the difference appears to be that in D. uninucleata the R-enantiomers are synthesized in mitochondria. Exogenously supplied eicosa pentaenoic acid was converted to 3-hydroxy 5Z,11Z,14Z,17Z-eicosapentaenoic acid by cell-free enzyme extracts though there was no requirement for a 5Z, 8Z-diene structure for the biosynthesis of 3-hydroxylated fatty acids as 3- hydroxy-8Z,11Z, 14Z, and 3-hydroxy-11Z,14Z,17Z-eicosatrienoic acids were sy nthesized from the corresponding fatty acids. We found no evidence for the synthesis of the prostaglandins F-2 alpha and E-2.