Structural and functional analysis and its application of the yeast novel N-asetylransferase that detoxifies a proline analogue

脯氨酸类似物解毒酵母新型N-乙酰基转移酶的结构和功能分析及其应用

• 批准号: 15380076
• 负责人: TAKAGI Hiroshi
• 金额: $ 9.54万
• 依托单位: Fukui Prefectural University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15380076/
• 关键词: 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

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 … More 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

在Σ1278b菌株中发现的出芽酵母Saccharomyces cerevisiae的MPR1基因（用于脯氨酸类似物抗性的sigma 1278b基因）编码一种新的n -乙酰基转移酶（MPR1），该酶可以解毒脯氨酸类似物azetidine-2-carboxylate （AZC）。在本研究中，我们分析了Mpr1的结构和功能如下。1)研究发现，mpr1破坏后的细胞对H_2O_2和热休克等氧化应激异常敏感，活性氧（ROS）水平升高。相反，MPR1的表达导致氧化处理后ROS水平降低，细胞活力增加。这些结果表明Mpr1可以降低细胞内ROS水平。研究还发现，put2中断的酵母细胞缺乏Δ^1-吡咯-5-羧酸（P5C）脱氢酶，ROS水平升高，而表达MPR1的put2中断的细胞ROS水平显著降低。Mpr1乙酰化P5C或其平衡化合物谷氨酸-γ-半醛。这些结果…More表明P5C对酵母细胞具有毒性是由于ROS的形成，而Mpr1在P5C诱导的氧化应激下调节ROS水平。2)我们发现酵母细胞在冷冻和解冻过程中ROS水平升高。基因破坏和表达实验表明，Mpr1通过降低细胞内ROS水平来保护酵母细胞免受冰冻胁迫。乙醇胁迫也得到了类似的结果。乙醇胁迫也得到了类似的结果。基因破坏和表达实验表明，Mpr1通过降低细胞内ros来保护酵母细胞免受乙醇胁迫。3)我们研究了Mpr1是否在细菌和植物细胞中具有类似的功能，以及它的表达是否可以赋予对AZC的抗性。我们的研究证实，MPR1可以在大肠杆菌和烟草中以类似于酵母的方式解毒有毒的AZC，因此可能被用作大肠杆菌和植物转化的新选择标记。综上所述，作为一种新的抗氧化机制，Mpr1可能通过乙酰化参与ROS生成的有毒代谢物（如P5C/GSA）来降低细胞内氧化水平。Mpr1在抗氧化应激中的作用有望用于培育新的抗应激酵母菌株。少

项目成果

Xing-Hai Zhang, H.Takagi, Jack.M.Widholm: "Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation"Plant Cell Reports. Vol.22 No.8. 615-622

张星海、H.Takagi、Jack.M.Widholm：“表达一种新型酵母基因，可以解毒脯氨酸类似物氮杂环丁烷-2-羧酸酯，从而在烟草种子发芽、愈伤组织和芽形成过程中赋予抗性”《植物细胞报告》。

Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation

• DOI: 10.1007/s00299-003-0741-3
• 发表时间: 2004-03-01
• 期刊: PLANT CELL REPORTS
• 影响因子: 6.2
• 作者: Zhang, XH;Takagi, H;Widholm, JM
• 通讯作者: Widholm, JM

酵母に発見した「アセチル化酵素Mpr1」による新しい抗酸化メカニズム活性酸素種の生成に関わる物質を基質とした酸化ストレス防御システム

在酵母中发现的使用乙酰转移酶 Mpr1 的新抗氧化机制 使用参与活性氧生成的物质作为底物的氧化应激防御系统

• 发表时间: 2006
• 期刊: 化学と生物 44
• 作者: Yusei Miyamoto;Atsuhiro Kanayama;Jun-Ichiro Inoue;Yoshiko Sugita-Konishi Makoto Shimizu.;高木 博史
• 通讯作者: 高木 博史

健康長寿研究成果報告書 酵母に見いだした酵素Mpr1よる新しい抗酸化メカニズム

健康长寿研究成果报告：酵母中发现Mpr1酶新抗氧化机制

• 发表时间: 2005
• 作者: M.Nomura;H.Takagi;高木 博史(分担執筆)
• 通讯作者: 高木 博史(分担執筆)

N-acetyltransferase Mpr1 confers freeze tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species

• DOI: 10.1093/jb/mvi134
• 发表时间: 2005-10-01
• 期刊: JOURNAL OF BIOCHEMISTRY
• 影响因子: 2.7
• 作者: Du, XY;Takagi, H
• 通讯作者: Takagi, H