Characterization of hydroxybenzoate prenyltransferase via molecular biological approach
| Project/Area Number |
17310126
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | Kyoto University |
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Principal Investigator |
YAZAKI Kazufumi Kyoto University, Research Institute for Sustainable Humanosphere, Professor (00191099)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,750,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥1,350,000)
Fiscal Year 2007: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
Fiscal Year 2006: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2005: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Keywords | hydroxybenzoic acid / prenyltransferase / membrane protein / secondary metabolism / shikonin / ubiquinone / biosynthesis / three dimensional model |
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| Research Abstract |
The aromatic substrate prenyltransferase (AS-PT) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane a-helices. Para-hydroxybenzoic acid prenyltransferases (PPTs) are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in this study as representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in E. coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus, the substrate specificity was analyzed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A three dimensional (3-D) molecular model of the substrate-binding site consistent with these biochemical findings was generated.
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Report
(4 results)
Research Products
(54 results)
