Marine algae are a major source of ω-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae requires the desaturation of fatty acids by Δ6-desaturases, and enzymes from different species display a range of specificities toward ω3- and ω6-LCPUFA precursors. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b₅ domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This data set provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.

中文翻译：

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共有突变和祖先重建提供洞察底物特异性和前端Δ6-去饱和酶家族的演变

海藻是ω-3长链多不饱和脂肪酸（ω3-LCPUFAs）的主要来源，ω-3LCPUFAs是人类有条件的必需营养素，是工业生产的目标。这些分子在海藻中的生物合成需要通过Δ6-去饱和酶对脂肪酸进行去饱和，并且来自不同物种的酶对ω3-和ω6-LCPUFA前体表现出一系列特异性。在没有分子结构的情况下，对Δ6-去饱和酶的可变底物特异性的结构基础了解甚少。在这里，我们对Δ6-去饱和酶家族进行了诱变和祖先蛋白质重建的共识分析，重点研究了来自Micromonas pusilla的ω3-特异性Δ6-去饱和酶（MpΔ6des）和来自双孢子菌（ω3/ω6）的双特异性（ω3/ω6）Δ6-去饱和酶。葡萄球菌（OtΔ6des）。我们对MpΔ6des中共有氨基酸取代的表征表明，该蛋白质不同区域的残基，例如N端细胞色素b ₅结构域，可以对确定底物特异性做出重要贡献。祖先蛋白质的重建还表明，某些现有的Δ6-去饱和酶，例如OtΔ6des，可能会因失去对ω3-LCPUFAs的特异性而适应不同的环境条件。该数据集提供了Δ6-去饱和酶中有助于底物特异性的区域图，并可能有助于将来尝试改造这些蛋白质以用于生物技术。