title: Biosynthesis of C18 polyunsaturated fatty acids in microsomal membrane preparations from the filamentous fungus Mucor circinelloides.
abstract: The biosynthesis of C18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained delta9, delta12 and delta6 desaturase activities. The delta9 desaturase exhibited characteristics similar to those of the animal and yeast delta9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate. Cytochrome b5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH:cytochrome b5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time-course studies with radiolabelled oleoyl-CoA showed that the oleate [18:1(9)] was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho. Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the delta12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho. Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn-1 and sn-2 positions served as substrate for the delta12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine:acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. Delta6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant. Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate [18:2(9,12)] and gamma-linolenate [18:3(6,9,12)] on this phospholipid is similar to that reported for oil seed membranes.
authors.affiliation: School of Biological Sciences, University of Bristol, UK.
authors.forename: F M
authors.initials: FM
authors.lastname: Jackson
authors.forename: T C
authors.initials: TC
authors.lastname: Fraser
authors.forename: M A
authors.initials: MA
authors.lastname: Smith
authors.forename: C
authors.initials: C
authors.lastname: Lazarus
authors.forename: A K
authors.initials: AK
authors.lastname: Stobart
authors.forename: G
authors.initials: G
authors.lastname: Griffiths
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D015786:Cytochromes b5 / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000032:analysis / Q000096:biosynthesis*; D006168:Guinea Pigs; D006368:Helianthus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D008861:Microsomes / Q000378:metabolism*; D009089:Mucor / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D005231:Fatty Acids, Unsaturated; C017725:stearoyl-coenzyme A; D015786:Cytochromes b5; D044943:Fatty Acid Desaturases; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase