Anglysis of novel gevefor proline analogue resistance found in budding yesst Σ12786

新兴yesst Σ12786中发现的新型gevefor脯氨酸类似物抗性分析

• 批准号: 12660084
• 负责人: TAKAGI Hiroshi
• 金额: $ 2.11万
• 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 (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660084/
• 关键词: budding yeast; N-acetyltransferase; Prline analogue resistance; azetidine-2-carboxylic acid; genome project; 転写調節因子; パーミアーゼ; L-アゼチジン-2-カルボン酸

We recently have discovered, on the chromosome of the budding yeast Saccharmyces cerevisiae Σ1278b, a novel gene MPR1 (sigma 1278b gene for L-proline-analogue resistance) required for the resistance of Σ1278 backgraound strains to toxic AZC.1. The genes MPR1 and MPR2, both characterized by one amino acid change at position 85, were present on chromosomes XIV and X of Σ1278b background strains, respectively, but were absent in the S. cerevisiae S288C used for the genomic sequence determination.2. Gene expression in Escherichia coli and enzymatic analysis showed that MPR1 encodes a novel AZC acetyltransferase, by which L-proline itself and other L-proline analogues are not acetylated. The MPR1-encoded protein (Mprlp) was considered to be a member of the N-acetyltansferase superfamily. We believe that AZC is converted to N-acetyl AZC by Mprlp in the cytoplasm, and consequently, N-acetyl AZC no longer replaces L-proline during the biosynthesis of protein.3. By the comparative analysis of MPR1 in the S. cerevisiae complex species, the type strain of S. paradoxus exhibited resistance and acetyltransferase activity to AZC. The new MPR1 homologue (Spa MPR1) from S. paradoxus was isolated by PCR with the primers based on the sequence of MPR1. Gene expression and enzymatic analysis showed that the cloned Spa MPR1 encodes an AZC acetyltransferase, which has 87% identity to Mprlp.4. The amino acid sequence of the predicted Mprlp was homologous to that of the fission yeast Schizosaccharomyces pombe hypothetical 23.8 kDa protein. We analyzed the S. pombe homologue of MPR1, ppr1^+ (pombe MPR1). This gene was responsibel to the AZC resistance of S. pombe and encodes an acetyltransferase that detoxifies AZC in a manner similar to that by MPR1.Our findings here suggest that this enzyme may have a physiologically conserved function additional to that of detoxifying unusual imino acid AZC.

我们最近在出芽酵母Saccharmyces cerevisiae Σ1278b的染色体上发现了一个新的基因MPR1 （l -脯氨酸类似物抗性的sigma 1278b基因），该基因是Σ1278背景菌株对有毒AZC.1的抗性所必需的。MPR1和MPR2基因分别存在于Σ1278b背景菌株的XIV和X染色体上，其85位上都有一个氨基酸的变化，但在用于基因组序列测定的酿酒酵母S288C中不存在。基因在大肠杆菌中的表达和酶促分析表明，MPR1编码一种新的AZC乙酰转移酶，该酶可使l -脯氨酸和其他l -脯氨酸类似物不被乙酰化。mpr1编码蛋白（Mprlp）被认为是n -乙酰转移酶超家族的成员。我们认为AZC在细胞质中被Mprlp转化为n -乙酰基AZC，因此在蛋白质的生物合成过程中n -乙酰基AZC不再取代l -脯氨酸。通过对酿酒葡萄球菌复合体种中MPR1的比较分析，发现悖论葡萄球菌型菌株对AZC具有抗性和乙酰转移酶活性。利用引物对悖论葡萄球菌的MPR1序列进行PCR分离，获得了新的同源物（Spa MPR1）。基因表达和酶学分析表明，克隆的Spa MPR1编码AZC乙酰转移酶，与MPR1的同源性为87%。预测的Mprlp的氨基酸序列与裂变酵母Schizosaccharomyces pombe假设的23.8 kDa蛋白的氨基酸序列同源。我们分析了MPR1的S. pombe同源物ppr1^+ (pombe MPR1)。该基因与S. pombe的AZC抗性有关，并编码一种乙酰转移酶，该酶以与MPR1相似的方式解毒AZC。我们的研究结果表明，这种酶除了解毒不寻常的亚胺酸AZC外，还可能具有生理上保守的功能。

项目成果

Hiroshi Takagi, Mika Shichiri, Miho Takemura, Miho Mohri, Shigeru Nakamori: "Saccharomyces cerevisiae Σ1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance"Journal of Bacteriology. 182. 4249-4256 (20

Hiroshi Takagi、Mika Shichiri、Miho Takemura、Miho Mohri、Shigeru Nakamori：“酿酒酵母 Σ1278b 具有 L-脯氨酸类似物抗性所需的 N-乙酰转移酶基因超家族的新基因”《细菌学杂志》182. 4249-4256 (20)

Michiyo Nomura, Shigeru Nakamori, Hiroshi Takagi: "Characterization of novel acetyltransferases found in the budding and fission yeasts that detoxifies a proline analogue, azetidine-2-carboxylic acid"Journal of Biochemistry. 133. 67-74 (2003)

Michiyo Nomura、Shigeru Nakamori、Hiroshi Takagi：“在芽殖酵母和裂殖酵母中发现的新型乙酰转移酶的特征，该酶可以解毒脯氨酸类似物氮杂环丁烷-2-羧酸”《生物化学杂志》。

Yasuko Kimura, Shigeru Nakamori and Hiroshi Takagi: "Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species"Yeast. 19. 1437-1445 (2002)

Yasuko Kimura、Shigeru Nakamori 和 Hiroshi Takagi：“酿酒酵母复杂物种中有毒脯氨酸类似物抗性所需的 MPR1 基因的多态性”。

高木博史: ""General Interest"への抵抗"バイオサイエンスとインダストリー. 60. 472 (2002)

Hiroshi Takagi：“抵制‘普遍利益’”《生物科学与工业》60. 472 (2002)。

Mika Shichiri, Chikara Hoshikawa, Shigeru Nakamori and Hiroshi Takagi: "A novel acetyltranferase found in Saccharomyces cerevisiae Σ1278b that detoxifies a proline analogue, azetidine-2-carboxylic acid"Journal of Biological Chemistry. 276. 41998-42002 (20

Mika Shichiri、Chikara Hoshikawa、Shigeru Nakamori 和 Hiroshi Takagi：“在酿酒酵母 Σ1278b 中发现的一种新型乙酰转移酶，可以解毒脯氨酸类似物氮杂环丁烷-2-羧酸”《生物化学杂志》276。41998-42002（20）