| Project/Area Number |
18570137
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Nippon Medical School |
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Principal Investigator |
NISHINO Tomoko Nippon Medical School, Department of Biochemistry and Molecular Biology, Senior Assistant Professor (80075613)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | xantine oxidase / xanthine dehvdroeenase / flavoenzvme / molybdenum enzyme / reactive oxveen sued / insect cell expression system / crystal structure / dehydrogenase-oxidase conversion |
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| Research Abstract |
Xanthine oxidoreductase (XOR) is a complex metalloflavoenzyme of protein of Mr 300kDa and are composed of two identical subunits and independent subunits; each subunit contains ome molybdopterin, two non-identical Fe2S2 centers, and flavin adenin dinucleotide. Xanthine oxidoreductase exists originally as a dehydrogenase using NAD^+ as electron acceptor, but only the mammalian enzyme converts to oxidase form using oxygen as a electron acceptor We showed the mechanism of xantine dehydrogenase to oxidase conversion in recent studies. Recent years, reactive oxygen species (ROS) are earnestly studying as important intra-cellular signal transferring molecules. Xanthine oxidoreductase is one of the important enzymes of ROS-prcducible system. Based on the structural differences between the bovine dehydrogenase and oxidase and site-directed mutagenesis studies of rat XOR expressed in baculovirus Sf9/incect cell system, we identified two disulfide bonds for reversible conversion as Cys535/Cys992
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and Cys1316/Cys1324. The formation of disulfide bonds worked as trigger to disrupt the unique amino acids cluster (Arg334/Trp335/Arg426/Phe549) situated re-face of FAD following the invasion of active long loop of Gln422-Lys432 into NAD^+ binding root. So disulfide bonds formation finally caused dehydrogenase to oxidase conversion. The cluster disruption largely changed the ionic environment around FAD and this phenomenon also influenced on the highly reactivity of FAD to oxygen. Based on the structural mechanism of dehydrogenase to oxidase conversion, we constructed the xanthine oxidase mutant of W335A/F336L that amino acids cluster was disrupted and at the same time redox potential of FAD elevated like oxidase form. This mutant expressed as always oxidase form even after treated with DTT. The crystal structure of this mutant has been determined at 2.3 A resolution. This mutant produced large amount of ROS (O_2・) . The percentage of one-electron transfer to O_2 with the DTT-treated non-treated mutant was~75% and the value was much higher than that of native enzyme from rat liver: the values with native enzyme were~35% and~15% for DTT-treated untreated enzyme respectively. The mutant was thought to be a good model of highly superoxide-productive enzyme system. In 2007, we constructed the transgenic mouse of double mutated W335A/F336L XOR instead of wild type XOR. We will plan to collect the data of lives of transgenic mice and investigate the pathological evidences and biochemical data of various organs in transgenic mice. Less
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