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ANALYSIS OF THE GENES ENCODING UBIQUINONE BIOSYNTHETIC ENZYMES FROM HIGHER EUKARYOTES

高等真核生物泛醌生物合成酶编码基因的分析

基本信息

批准号：
16380073
负责人：
KAWAMUKAI Makoto
金额：
$ 7.3万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16380073/
关键词：
UBIQUINONE COQ ANTIOXIDANT FISSION YEAST RESPIRATION PRENYL TRANSFERASE

项目摘要

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.

泛醌（Q）的类异戊二烯链由微生物和哺乳动物中的反式异戊二烯基二磷酸合酶决定。由于小鼠和人类产生Q-9和Q-10，预期它们将茄呢基和癸烯基二磷酸脱氢酶作为一种泛醌的决定酶。在这里，我们表明，小鼠和人类茄呢基和十异戊二烯基二磷酸脱氢酶是由新表征的hDPS 1（mSPS 1）和hDLP 1（mDLP 1），这被确定为直向同源物的粟酒裂殖酵母Dps 1和Dlp 1，分别异源四聚体。而hDPS 1和mSPS 1可以作为S. pombe dps 1破坏剂，hDLP 1和mDLP 1均不能与S. pombe dlp 1破坏剂。因此，只有hDPS 1和mSPS 1是SpDps 1的功能性直系同源物。大肠杆菌工程表达鼠和人SpDps 1和/或SpDlp 1同源物和它们的泛醌类型进行了测定。而表达单一组分的转化子仅从E. coli来源，表达mSPS 1和mDLP 1或hDPS 1和hDLP 1的双转化体分别产生Q-9或Q-10，并验证了茄呢基或十异戊二烯基二磷酸合酶的体外活性。人和鼠的长链transrenyl二磷酸脱氢酶的复杂的大小，估计通过凝胶过滤色谱法，表明它们由异四聚体。在大肠杆菌中的表达大肠杆菌的异源组合，即mSPS 1和hDLP 1或hDPS 1和mDLP 1，产生Q-9和Q-10，表明这两种组分都参与决定泛醌侧链。因此，我们确定了在哺乳动物中确定泛醌侧链的酶的组分。