Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-09-06 DOI:10.1016/j.pep.2024.106604

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引用次数: 0

Abstract

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.

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来自 Rahnella aquatilis 的新型 L-isoleucine-4-dioxygenase (RaIDO) 的生物化学和结构特征。

L-异亮氨酸-4-二加氧酶可将 L-异亮氨酸（Ile）转化为（2S,3R,4S）-4-（OH）-异亮氨酸（4-HIL），这是一种天然存在的羟基氨基酸，是一种很有希望用于药物和功能性食品开发的化合物。本文克隆、表达并鉴定了一种新型 L-异亮氨酸-4-二加氧酶（RaIDO），它是仅有的几种报道的 L-异亮氨酸-4-二加氧酶之一。以 Ile 为底物时，RaIDO 表现出较高的催化效率和良好的稳定性。HPLC-MS 和 NMR 证实，RaIDO 能将 Ile 转化为 (2S,3R,4S)-4-(OH)-异亮氨酸。此外，RaIDO的结构分析还揭示了关键的活性位点残基，包括H159、D161和H212。RaIDO 酶的最佳反应温度范围为 30°C-45°C，在 40°C 时催化活性最高。此外，该酶的最佳 pH 值为 8.0。因此，新型 L-异亮氨酸-4-二加氧酶（RaIDO）具有催化效率高和稳定性好的特点，是工业应用的理想选择。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

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.