Metabolism of 18:2( n − 6), 18:3( n − 3), 20:4( n − 6) and 20:5( n − 3) by the fungus Gaeumannomyces graminis: Identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation

Abstract

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2( n − 6)), linolenic acid (18:3( n − 3)), arachidonic acid (20:4( n − 6)) and eicosapentaenoic acid (20:5( n − 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2( n − 6) was metabolized mainly to 8-hydroxy-9,12-octade-cadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (−)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3( n − 3) was converted to 8-hydroxy-9,12,15-oc-tadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4( n − 6) or 20:5( n − 3) could not be detected. 20:4( n − 6) was efficiently converted to 18( R)-hydroxyeicosatetraenoic acid (18( R)-HETE) and 19( R-HETE and to traces of 17-HETE, while 20:5( n − 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.