Jackson F M, Fraser T C, Smith M A, Lazarus C, Stobart A K, Griffiths G
School of Biological Sciences, University of Bristol, UK.
Eur J Biochem. 1998 Mar 15;252(3):513-9. doi: 10.1046/j.1432-1327.1998.2520513.x.
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.
已对卷枝毛霉中C18多不饱和脂肪酸的生物合成进行了研究。微粒体膜制剂含有Δ9、Δ12和Δ6去饱和酶活性。Δ9去饱和酶表现出与动物和酵母Δ9去饱和酶相似的特性，即与膜结合并利用硬脂酰辅酶A作为底物。细胞色素b5（一种缺乏20个氨基酸疏水C末端的可溶性形式）刺激去饱和作用，并被鉴定为膜的主要细胞色素成分。高铁氰化物还原酶活性高（表明NADH：细胞色素b5还原酶活性）且受氰化物抑制，这进一步支持了其与哺乳动物和酵母酶的相似性。用放射性标记的油酰辅酶A进行的时间进程研究表明，油酸[18:1(9)]转移到磷脂酰胆碱（PtdCho）的sn-2位，并去饱和生成亚油酰-PtdCho。在添加还原剂之前从膜中去除过量的油酰辅酶A，证实油酰-PtdCho是Δ12去饱和酶的底物。溶血磷脂酰胆碱：酰基辅酶A酰基转移酶（LPCAT）的活性促进了油酸在磷脂这个位置的进入，该酶很容易将油酸从油酰辅酶A转移到溶血磷脂酰胆碱上。在油酸通过肯尼迪途径的酶进入磷脂后，也证明了PtdCho的sn-1位上的油酸去饱和。因此，sn-1和sn-2位上的油酸作为Δ12去饱和酶的底物，这与在油料种子组织中的观察结果一致。LPCAT活性显著高于溶血磷脂酰乙醇胺：酰基辅酶A酰基转移酶（LPEAT）的活性，这表明油酸较少有效地被导向磷脂酰乙醇胺用于亚油酸合成。未证明磷脂酰肌醇上有去饱和作用。Δ6去饱和酶在有还原剂存在的情况下，利用外源提供的PtdCho的sn-2位上的亚油酸作为底物。因此，底物通过LPCAT进入PtdCho以及在这种磷脂上合成亚油酸[18:2(9,12)]和γ-亚麻酸[18:3(6,9,12)]与油料种子膜的情况相似。
