{"title":"来自 Rahnella aquatilis 的新型 L-isoleucine-4-dioxygenase (RaIDO) 的生物化学和结构特征。","authors":"","doi":"10.1016/j.pep.2024.106604","DOIUrl":null,"url":null,"abstract":"<div><p>The <sub>L</sub>-isoleucine-4-dioxygenase converts <sub>L</sub>-isoleucine (Ile) into(2<em>S</em>,3<em>R</em>,4<em>S</em>)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel <sub>L</sub>-isoleucine-4-dioxygenase (<em>Ra</em>IDO) from <em>Rahnella aquatilis</em> was cloned, expressed and characterized, as one of only a few reported <sub>L</sub>-isoleucine-4-dioxygenases. <em>Ra</em>IDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that <em>Ra</em>IDO converts Ile into (2<em>S</em>,3<em>R</em>,4<em>S</em>)-4-(OH)-isoleucine. Further, structural analysis of <em>Ra</em>IDO revealed key active site residues, including H159, D161 and H212. The <em>Ra</em>IDO enzyme showed an optimal reaction temperature range of 30°C–45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel <sub>L</sub>-isoleucine-4-dioxygenase (<em>Ra</em>IDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4000,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis\",\"authors\":\"\",\"doi\":\"10.1016/j.pep.2024.106604\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The <sub>L</sub>-isoleucine-4-dioxygenase converts <sub>L</sub>-isoleucine (Ile) into(2<em>S</em>,3<em>R</em>,4<em>S</em>)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel <sub>L</sub>-isoleucine-4-dioxygenase (<em>Ra</em>IDO) from <em>Rahnella aquatilis</em> was cloned, expressed and characterized, as one of only a few reported <sub>L</sub>-isoleucine-4-dioxygenases. <em>Ra</em>IDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that <em>Ra</em>IDO converts Ile into (2<em>S</em>,3<em>R</em>,4<em>S</em>)-4-(OH)-isoleucine. Further, structural analysis of <em>Ra</em>IDO revealed key active site residues, including H159, D161 and H212. The <em>Ra</em>IDO enzyme showed an optimal reaction temperature range of 30°C–45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel <sub>L</sub>-isoleucine-4-dioxygenase (<em>Ra</em>IDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.</p></div>\",\"PeriodicalId\":20757,\"journal\":{\"name\":\"Protein expression and purification\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.4000,\"publicationDate\":\"2024-09-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Protein expression and purification\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1046592824001761\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592824001761","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis
The L-isoleucine-4-dioxygenase converts L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C–45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.
期刊介绍：
Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.
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