| Budget Amount *help |
¥11,400,000 (Direct Cost: ¥11,400,000)
Fiscal Year 2006: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2005: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2004: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Research Abstract |
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.
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