ANALYSIS OF THE GENES ENCODING UBIQUINONE BIOSYNTHETIC ENZYMES FROM HIGHER EUKARYOTES

泛醌(Q)的类异戊二烯链是由微生物和哺乳动物的转聚二磷酸合成酶决定的。当小鼠和人类产生Q-9和Q-10时，它们被认为具有茄氨酰基和癸烯酰二磷酸合成酶作为一种泛醌的决定酶。在这里，我们发现小鼠和人类的solanesyl和decaprenyl二磷酸合成酶是由新发现的hDPS1 （mSPS1）和hDLP1 （mDLP1）组成的异源四聚体，它们分别被鉴定为S.pombe Dps1和Dlp1的同源物。虽然hDPS1或mSPS1可以补充S. pombe dps1干扰物，但hDLP1和mDLP1都不能补充S. pombe dlp1干扰物。因此，只有hDPS1和mSPS1是SpDps1的功能同源物。利用大肠杆菌表达小鼠和人SpDps1和/或SpDlp1同源物，并测定其泛素类型。表达单一组分的转化子仅从大肠杆菌中产生Q-8，而表达mSPS1和mDLP1或hDPS1和hDLP1的双转化子分别产生Q-9或Q-10，并验证了茄氨酰或十烯丙烯酰二磷酸合成酶的体外活性。凝胶过滤色谱法测定了人和鼠长链透明二磷酸酯合成酶的复合物大小，表明它们是由异源四聚体组成的。在大肠杆菌中表达异种组合，即mSPS1和hDLP1或hDPS1和mDLP1，产生Q-9和Q-10，表明这两个组分都参与了泛醌侧链的确定。因此，我们确定了决定哺乳动物中泛素侧链的酶的成分。

The isoprenoid chain of ubiquinone (Q) is determined by transpolyprenyl diphosphate synthase in microorganisms and presumably in mammals. As mice and humans produce Q-9 and Q-10, they are expected to posses solanesyl and decaprenyl diphosphate synthases as the determining enzyme for a type of ubiquinone. Here we show that murine and human solanesyl and decaprenyl diphosphate synthases are heterotetramers composed of newly characterized hDPS1 (mSPS1) and hDLP1 (mDLP1), which were identified as orthologs of the S.pombe Dps1 and Dlp1, respectively. While hDPS1 or mSPS1 can complement the S. pombe dps1 disruptant, neither hDLP1 nor mDLP1 could complement the S. pombe dlp1 disruptant. Thus, only hDPS1 and mSPS1 are functional orthologs of SpDps1. Escherichia coli was engineered to express the murine and human SpDps1 and/or SpDlp1 homologues and their ubiquinone types were determined. While transformants expressing a single component only produced Q-8 from E. coli origin, double transformants expressing mSPS1 and mDLP1 or hDPS1 and hDLP1 produced Q-9 or Q-10, respectively, and an in vitro activity of solanesyl or decaprenyl diphosphate synthase was verified. The complex size of the human and murine long-chain transprenyl diphosphate synthases, as estimated by gel filtration chromatography, indicates that they consist of heterotetramers. Expression in E. coli of heterologous combinations, namely, mSPS1 and hDLP1 or hDPS1 and mDLP1, generated both Q-9 and Q-10, indicating both components are involved in determining the ubiquinone side chain. Thus, we identified the components of the enzymes that determine the side chain of ubiquinone in mammals.