Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina; and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Esmeralda y Ocampo, Rosario, Argentina.
Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina; and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Esmeralda y Ocampo, Rosario, Argentina
J Lipid Res. 2018 Oct;59(10):1871-1879. doi: 10.1194/jlr.M085258. Epub 2018 Aug 7.
Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of DesA desaturase improves Des activity. In addition, the replacement of the first two transmembrane domains from inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipid-water interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of Δ5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes.
关于膜结合脂质去饱和酶的结构-功能关系知之甚少。我们使用结构域交换策略发现，DesA 去饱和酶的 N 端（包含前两个跨膜片段）区域可提高 Des 活性。此外，用 DesA 相应的结构域替换无活性开放阅读框（ORF）BL02692 的前两个跨膜结构域足以复活该酶。出乎意料的是，我们能够通过在靠近脂水界面的第一个跨膜结构域中定位的半胱氨酸（Cys40Tyr）的单个取代来恢复 ORF BL02692 的活性。用定点诱变取代八个残基（Gly90、Trp104、Lys172、His228、Pro257、Leu275、Tyr282 和 Leu284）产生了 DesA 的无活性变体。DesA 的同源建模表明 His228 是金属结合中心的一部分，与典型的 His 框一起。Trp104 形成疏水性隧道，而 Gly90 和 Lys172 可能参与底物结合/识别。Pro257、Leu275、Tyr282 和 Leu284 可能与活性位点的结构排列或与电子供体的相互作用有关。这项研究揭示了 Δ5 磷脂去饱和酶的 N 端区域以及该酶类活性所需的单个残基的作用。
