Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement

IF 4.5 2区医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Bioorganic Chemistry Pub Date : 2024-08-23 DOI:10.1016/j.bioorg.2024.107744

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Abstract

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 (k_cat/K_m) 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.

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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

底物通路隧道工程是酶修饰的一种有效策略。在本研究中，我们通过突变底物通道入口处的 Q86 残基，提高了荧光假单胞菌 ZJUT001（PfNHase）的铁型腈氢化酶（Fe-type NHase）的催化性能。与野生型 PfNHase（PfNHase-WT）相比，突变体 PfNHase-αQ86W 对苯甲腈、2-氰基吡啶、3-氰基吡啶和 4-羟基苯甲腈的催化活性分别提高了 9.35 倍、3.30 倍、6.55 倍和 2.71 倍。此外，突变体 PfNHase-αQ86W 对苯甲腈的催化效率（kcat/Km）是野生型 PfNHase-WT 的 17.32 倍。有趣的是，PfNHase-αQ86W 对底物的偏好从脂肪族腈类转移到了芳香族腈类底物。我们的分析深入研究了导致这种底物偏好改变的结构变化，强调了入口通道区域的扩大、底物结合袋的扩大以及底物与酶之间疏水相互作用的增加。分子动力学模拟和动态交叉相关矩阵（DCCM）进一步支持了这些发现，为增强对芳香族腈底物的催化活性提供了全面的解释。

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来源期刊

Bioorganic Chemistry 生物-生化与分子生物学

CiteScore

9.70

自引率

3.90%

发文量

679

审稿时长

31 days

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.