Structural and functional analysis and its application of the yeast novel N-asetylransferase that detoxifies a proline analogue
| Project/Area Number |
15380076
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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 |
Applied biochemistry
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| Research Institution | Fukui Prefectural University |
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Principal Investigator |
TAKAGI Hiroshi Fukui Prefectural University, Department of Bioscience, Professor, 生物資源学部, 教授 (50275088)
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| Co-Investigator(Kenkyū-buntansha) |
ODA Junichi Fukui Prefectural University, Department of Bioscience, Professor, 生物資源学部, 教授 (50027041)
HIBI Takao Fukui Prefectural University, Department of Bioscience, Associate Professor, 生物資源学部, 助教授 (00285181)
TAKAHASHI Masakazu Fukui Prefectural University, Department of Bioscience, Assistant Professor, 生物資源学部, 助手 (80315837)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2005: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2003: ¥7,000,000 (Direct Cost: ¥7,000,000)
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| Keywords | Yeast / N-Acetyltrasnferase / Azetidine-2-carboxylic acid / Anti-oxidant enzyme / Oxidative stress / Reactive oxygen species / Freezing stress / Ethanol stress / プロリンアナログ耐性 |
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| Research Abstract |
The MPR1 gene (sigma 1278b gene for proline-analogue resistance) of the budding yeast Saccharomyces cerevisiae, which is found in the Σ1278b strain, encodes a novel N-acetyltransferase (Mpr1) that detoxifies the proline analogue azetidine-2-carboxylate (AZC). In this study, we analyzed the structure and function of Mpr1 as follows.
1)We found that mpr1-disrupted cells were hypersensitive to oxidative stresses such as H_2O_2 and heat shock and contained increased levels of reactive oxygen species (ROS). In contrast, expression of MPR1 leads to a decrease in ROS level and an increase in cell viability after oxidative treatments. These results indicate that Mpr1 can reduce intracellular ROS levels. It was also found that put2-disrupted yeast cells lacking Δ^1-pyrroline-5-carboxylate (P5C) dehydrogenease increased ROS levels and put2-disrupted cells that expressed MPR1 had considerably lower ROS levels. Mpr1 acetylates P5C or its equilibrium compound glutamtate-γ-semialdehyde. These results
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suggest that P5C is toxic to yeast cells due to the formation of ROS and that Mpr1 regulates the ROS level under P5C-induced oxidative stress.
2) We found that yeast cells exhibited increased levels of ROS during freezing and thawing. Gene disruption and expression experiments indicated that Mpr1 protects yeast cells from freezing stress by reducing the intracellular levels of ROS. Similar results were obtained from ethanol stress. Similar results were obtained from ethanol stress. Gene disruption and expression experiments indicated that Mpr1 protect yeast cells from ethanol stress by reducing the intracellular ROS.
3) We investigated whether MPR1 would function similarly in bacterial and plant cells and if its expression could confer resistance to AZC. Our studies confirm that MPR1 can function in a similar fashion in Escherichia coli and tobacco as in yeast to detoxify the toxic AZC so could potentially be used as a new selectable marker for E.coli and plant transformation.
In summary, as a novel antioxidant mechanism, Mpr1 is supposed to reduce the intracellular oxidation level by acetylating the toxic metabolite(s) involved in ROS production, such as P5C/GSA. The proposed role of Mpr1 against oxidative stress could be promising for breeding novel stress-resistant yeast strains. Less
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Report
(4 results)
Research Products
(18 results)
