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

长链合成聚戊烯基二磷酸合成酶是合成泛醌的侧链酶类，目前对它的分析大多是从原核生物起源的，而对真核生物酶类的性质了解甚少。在这里，我们发现从裂糖菌中提取的十戊烯二磷酸合成酶与原核类型的酶不同，形成异源四聚体。我们发现新蛋白D1pl是Dps1的伴侣，并在pombe中形成一个异四聚体复合物，构成一个活性的十戊烯二磷酸合成酶。Dps1与其他戊烯基二磷酸合成酶高度同源，而Dlp1仅与Dps1保持弱同源性。dlp1的缺失突变体丧失了十烯丙烯二磷酸合成酶的酶活性，不产生UQ-10，导致了pombe典型的泛素减少表型，即对H_2O_2过敏，需要抗氧化剂在最小培养基上生长，H_2S产量增加。dps1和dlp1突变体都可以通过表达细菌驱动的单功能十烯丙烯二磷酸合成酶来恢复，这表明dps1和dlp1都是酶活性所必需的。此外，我们发现Dps1和Dlp1形成了一个异四聚体复合物，并在大肠杆菌的一种活性酶中重组。因此，来自真核生物的二磷酸十戊烯基构成了一种在原核生物中未发现的异四聚体型酶结构。

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.