{"title":"荧光假单胞菌 ZJUT001 的铁型腈水解酶的底物通道工程，用于调整底物偏好和提高催化性能","authors":"","doi":"10.1016/j.bioorg.2024.107744","DOIUrl":null,"url":null,"abstract":"<div><p>Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from <em>Pseudomonas fluorescens</em> ZJUT001 (<em>Pf</em>NHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant <em>Pf</em>NHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type <em>Pf</em>NHase (<em>Pf</em>NHase-WT). In addition, the mutant <em>Pf</em>NHase-αQ86W showed a catalytic efficiency (<em>k<sub>cat</sub>/K<sub>m</sub></em>) towards benzonitrile 17.32-fold higher than the <em>Pf</em>NHase-WT. Interestingly, the substrate preference of <em>Pf</em>NHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, the<!--> <!-->enlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.</p></div>","PeriodicalId":257,"journal":{"name":"Bioorganic Chemistry","volume":null,"pages":null},"PeriodicalIF":4.5000,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement\",\"authors\":\"\",\"doi\":\"10.1016/j.bioorg.2024.107744\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from <em>Pseudomonas fluorescens</em> ZJUT001 (<em>Pf</em>NHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant <em>Pf</em>NHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type <em>Pf</em>NHase (<em>Pf</em>NHase-WT). In addition, the mutant <em>Pf</em>NHase-αQ86W showed a catalytic efficiency (<em>k<sub>cat</sub>/K<sub>m</sub></em>) towards benzonitrile 17.32-fold higher than the <em>Pf</em>NHase-WT. Interestingly, the substrate preference of <em>Pf</em>NHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, the<!--> <!-->enlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.</p></div>\",\"PeriodicalId\":257,\"journal\":{\"name\":\"Bioorganic Chemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.5000,\"publicationDate\":\"2024-08-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bioorganic Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0045206824006497\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bioorganic Chemistry","FirstCategoryId":"92","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0045206824006497","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement
Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, the enlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.
期刊介绍：
Bioorganic Chemistry publishes research that addresses biological questions at the molecular level, using organic chemistry and principles of physical organic chemistry. The scope of the journal covers a range of topics at the organic chemistry-biology interface, including: enzyme catalysis, biotransformation and enzyme inhibition; nucleic acids chemistry; medicinal chemistry; natural product chemistry, natural product synthesis and natural product biosynthesis; antimicrobial agents; lipid and peptide chemistry; biophysical chemistry; biological probes; bio-orthogonal chemistry and biomimetic chemistry.
For manuscripts dealing with synthetic bioactive compounds, the Journal requires that the molecular target of the compounds described must be known, and must be demonstrated experimentally in the manuscript. For studies involving natural products, if the molecular target is unknown, some data beyond simple cell-based toxicity studies to provide insight into the mechanism of action is required. Studies supported by molecular docking are welcome, but must be supported by experimental data. The Journal does not consider manuscripts that are purely theoretical or computational in nature.
The Journal publishes regular articles, short communications and reviews. Reviews are normally invited by Editors or Editorial Board members. Authors of unsolicited reviews should first contact an Editor or Editorial Board member to determine whether the proposed article is within the scope of the Journal.
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