Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast
Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast
复制标题
DOI：
10.1074/jbc.m305990200
复制
发表时间：
2003-09-12
影响因子：
4.8
通讯作者：
Heinz, E
中科院分区：
文献类型：
--
作者：
Domergue, F;Abbadi, A;Heinz, E
超长链多不饱和脂肪酸对健康的益处以及在转基因油籽作物中生产它们的长期目标导致了编码参与其生物合成的去饱和酶和延伸酶的所有基因的克隆。编码的活性已通过异源表达在体内得到证实，但对这些途径中涉及的实际酰基底物知之甚少。使用来自不同生物体的 Delta6 延伸酶和前端去饱和酶，我们在酿酒酵母中重建了从外源提供的亚油酸中花生四烯酸的生物合成，以鉴定这些酰基载体。酰基辅酶 A 测量结果强烈表明，多不饱和脂肪酸生物合成中涉及的延伸步骤发生在酰基辅酶 A 池内。相反，对脂质的详细分析表明，当使用来自低等植物、真菌、蠕虫和藻类的 Delta5-和 Delta6-去饱和酶时，两个去饱和步骤（Delta5 和 Delta6）主要发生在磷脂酰胆碱的 sn-2 位点。这些 Delta6-去饱和酶对在该特定位置酰化的脂肪酸的特异性以及与酰基辅酶 A 池的限制性再平衡导致 γ-亚麻酸在磷脂酰胆碱的 sn-2 位上积累，并阻止酵母中有效的花生四烯酸生物合成。我们通过使用类似的实验方法证实，相比之下，人类 Delta6-去饱和酶使用亚油酰辅酶A作为底物，这导致后续延伸步骤的高效率。此外，我们报告 Delta12 去饱和酶对脂质极性头基或 sn 位置没有特异性。
The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a Delta6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (Delta5 and Delta6) occur predominantly at the sn-2 position of phosphatidylcholine when using Delta5- and Delta6-desaturases from lower plants, fungi, worms, and algae. The specificity of these Delta6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of gamma-linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human Delta6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that Delta12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.