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Study on the variety of synthesizing enzyme for isoprenoid side chain in ubiquinone

泛醌类异戊二烯侧链合成酶品种的研究

基本信息

批准号：
14560067
负责人：
KAWAMUKAI Makoto
金额：
$ 2.3万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560067/
关键词：
UBIQUINONE COQ ANTIOXIDANT FISSION YEAST RESPIRATION PRENYL TRANSFERASE 生合成

项目摘要

Long-chain producing polyprenyl diphosphate synthase, which is responsible for the side chain of ubiquinone, has been mostly analyzed from prokaryotic origins, and little is known about the nature of eukaryotic enzyme. Here, we show decaprenyl diphosphate synthase from Schizosaccharomyces pombe forms a heterotetramer unlike prokaryotic types of enzymes. We found the novel protein named D1pl is the partner of Dps1, and formed a heterotetrameric complex to constitute an active decaprenyl diphosphate synthase in S. pombe. While Dps1 is the highly homologous protein with other prenyl diphosphate synthases, Dlp1 only retains weak homology with Dps1. The deletion mutant of dlp1 lost the enzymatic activity of decaprenyl diphosphate synthase, did not produce UQ-10 and resulted in a typical ubiquinone less phenotype in S. pombe, i.e., hypersensitivity to H_2O_2, requirement of antioxidant to grow on the minimal medium, and an elevated production of H_2S. Both dps1 and dlp1 mutants can be restored by expressing bacterial driven decaprenyl diphosphate synthase which is solely functional, indicating both dps1 and dlp1 is required for the enzymatic activity. Furthermore, we show Dps1 and Dlp1 formed a heterotetrameric complex and reconstituted in an active enzyme in Escherichia coli. Thus, decaprenyl diphosphate from an eukaryotic origin constitutes a heterotetrameric type of enzyme structure which was not found in prokaryotes.

产生长链的聚异戊二烯二磷酸合酶负责泛醌的侧链，主要是从原核起源进行分析，而对真核酶的性质知之甚少。在这里，我们展示了来自粟酒裂殖酵母的十异戊二烯二磷酸合酶形成了与原核类型的酶不同的异四聚体。我们发现名为D1pl的新蛋白是Dps1的伴侣，并形成异四聚体复合物，构成粟酒裂殖酵母中活性的十异戊二烯二磷酸合酶。虽然 Dps1 是与其他异戊二烯基二磷酸合酶高度同源的蛋白质，但 Dlp1 与 Dps1 仅保留微弱的同源性。 dlp1缺失突变体失去了十异戊二烯二磷酸合酶的酶活性，不产生UQ-10，导致粟酒裂殖酵母典型的泛醌缺乏表型，即对H_2O_2过敏，需要抗氧化剂才能在基本培养基上生长，并且H_2S产量增加。 dps1 和 dlp1 突变体都可以通过表达细菌驱动的十异戊二烯基二磷酸合酶来恢复，该酶具有单独的功能，表明 dps1 和 dlp1 都是酶活性所必需的。此外，我们还发现 Dps1 和 Dlp1 形成异四聚体复合物，并在大肠杆菌的活性酶中重建。因此，来自真核生物来源的十异戊二烯基二磷酸构成了在原核生物中未发现的异四聚体类型的酶结构。