在糖化酶BcX的活性位点结合二莫达尔基质
Mahin Saberi1, Aleksandra Chikunova1, Fredj Ben Bdira1
1Leiden Institute of Chemistry, Leiden University, The Netherlands.
The FEBS journal
|August 26, 2024
概括
由于其大型的活性部位,Bacillus circulans xylanase (BcX) 呈现出复杂的基质结合. 多种结合方向和部位会影响西兰的降解,影响酶动力学.
科学领域:
- 酶学 是一种酶学.
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- 细菌循环菌的西兰酶 (BcX),一个糖化酶11家族的酶,通过保留双位移机制降解西兰.
- BcX具有一个大型的活性位点裂和六个子位点,表明可能存在多个基质结合模式.
- 之前的研究表明,由BcX.进行糖化键的过程性水解.
研究的目的:
- 通过使用各种西洛斯寡合物,研究Bacillus circulans xylanase (BcX) 的基质结合模式.
- 阐明BcX活性位点内的多种基质相互作用的结构和机制基础.
- 描述不同结合部位对酶活性和动力学的影响.
主要方法:
- 催化不活的BcX变体 (BcX E78Q) 结晶结构的确定,该变体与西洛酸复合.
- 核磁共振 (NMR) 定位使用不同长度的西洛寡合体来分析结合动态.
- 关键活性部位残留物的部位定向突变发生,以评估它们在基质结合和结构完整性中的作用.
主要成果:
- 晶体结构揭示了活性部位,二次部位和第三表面部位的西洛结合.
- 核磁共振定位显示了非线性化学转移轨迹,表明快速交换的多重结合方向 (微到毫秒时间尺度).
- 突变性研究发现Trp9和指区域对于紧密的甘氨酸结合部位至关重要;突变F125A和W71A导致了显著的结构重组.
结论:
- BcX通过多个结合方向和在不同的部位,包括活性部位和二次/表面部位,容纳西寡合物.
- 在甘氨酸和甘氨酸位点的结合相互作用在很大程度上是独立的,在二级位点的结合也是如此.
- 该酶的复杂结合行为,以低毫米分离常数为特征,对于其西兰降解机制至关重要.
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