来自Acetivibrio热细胞ATCC 27405的多模块重组内葡萄糖酶AtGH9C-CBM3A-CBM3B的结构阐明及其基质结合分析
Ardhendu Mandal1, Jebin Ahmed1, Shweta Singh2
1Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
International journal of biological macromolecules
|June 19, 2024
概括
这项研究揭示了来自Acetivibrio热细胞的GH9内葡萄糖酶的结构和动态特性,确定了其对纤维素的过程活性的关键特征. 该酶优先结合纤维,这表明纤维素降解的特定机制.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 酶学 是一种酶学.
背景情况:
- 甘氨酸化酶 (GH) 9细胞系表现出多种活动,包括内,外和过程内模式,这种变化的根本原因尚不清楚.
- 了解细胞酶活性的结构基础对于生物质转化和生物燃料生产中的应用至关重要.
研究的目的:
- 从结构上描述一个重组GH9内葡萄糖酶,AtGH9C-CBM3A-CBM3B,来自Acetivibrio热细胞.
- 阐明AtGH9C-CBM3A-CBM3B的构造,基质结合和动态特性,以了解其细胞酶活性.
主要方法:
- 再组合蛋白的表达和净化.
- 循环二元化 (CD) 光谱用于二次结构分析.
- 分子动力学 (MD) 模拟以评估结构稳定性和动力学.
- 分子对接以预测基质结合亲和力.
- 小角度X射线散射 (SAXS) 用于溶液结构分析.
主要成果:
- 模拟的AtGH9C-CBM3A-CBM3B的结构显示了一个 (α/α) 6-桶折叠与催化三合一 (Asp98,Asp101,Glu489).
- 磁盘光谱显示了二次结构组成:25.2%的α-螺旋,18.4%的β-片,和56.4%的随机线圈.
- MD模拟显示结构稳定性,与结合的纤维糖导致较大的紧度 (RMSD 1.5 nm) 与未结合的酶 (RMSD 1.8 nm) 相比.
- 对接研究确定纤维为最佳基质 (ΔG = -5.05 kcal/mol),对较短或较长的纤维素链的亲和力降低.
- 该酶表现出过程性活性,循环3 (342-379 aa) 可能引导纤维素链结合和释放纤维.
- 萨克斯分析证实了溶液中的单分散的拳肘形结构,负的泽塔电位 (-24 mV) 表明稳定性和没有聚合.
结论:
- AtGH9C-CBM3A-CBM3B具有稳定的结构,并表现出过程性内葡萄糖酶活性,首选作用于细胞.
- CBM3B域在基质结合方面似乎不那么重要,而循环3在过程性方面起着重要作用.
- 该酶在溶液中的稳定性表明,在纤维素水解中有可能有生物技术应用.
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