Chen Anqi, Cheng Yuhan, Meng Liushi, Chen Jian
Science Center for Future Foods, Jiangnan University, Wuxi 214122, China.
Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
J Fungi (Basel). 2024 Nov 11;10(11):781. doi: 10.3390/jof10110781.
Trehalose is crucial for the stress resistance of , primarily through its stabilization of proteins and membranes. The Agt1 transporter, a member of the Major Facilitator Superfamily, mediates trehalose uptake, a key process for maintaining cellular integrity under stress. Despite its importance, the molecular mechanisms of Agt1-mediated trehalose transport remain underexplored. In this study, we expressed and purified the trehalase enzyme TreA from to develop reliable trehalose assays. We screened 257 wild isolates, identifying strains with enhanced trehalose transport capacities. Comparative analyses, including structural modeling and molecular docking, revealed that specific Agt1 variants exhibited significantly higher transport efficiency, influenced by key residues in the transporter. Molecular dynamics simulations and steered molecular dynamics provided further insights, particularly into the role of the Agt1 channel head region in substrate recognition and binding. Site-directed mutagenesis validated these findings, showing that mutations at critical residues, such as 156Q, 164L, 256Q, 395E, 396R, and 507Y significantly reduced transport activity, while 137Q, 230T, and 514 N increased efficiency under certain conditions.
海藻糖对于（此处原文缺失具体主体）的抗逆性至关重要，主要是通过其对蛋白质和膜的稳定作用。Agt1转运蛋白是主要易化子超家族的成员，介导海藻糖摄取，这是在应激条件下维持细胞完整性的关键过程。尽管其很重要，但Agt1介导的海藻糖转运的分子机制仍未得到充分探索。在本研究中，我们表达并纯化了来自（此处原文缺失具体来源）的海藻糖酶TreA，以开发可靠的海藻糖检测方法。我们筛选了257株野生（此处原文缺失具体类型）分离株，鉴定出具有增强海藻糖转运能力的菌株。包括结构建模和分子对接在内的比较分析表明，特定的Agt1变体表现出显著更高的转运效率，这受到转运蛋白中关键残基的影响。分子动力学模拟和引导分子动力学提供了进一步的见解，特别是关于Agt1通道头部区域在底物识别和结合中的作用。定点诱变验证了这些发现，表明关键残基如156Q、164L、256Q、395E、396R和507Y处的突变显著降低了转运活性，而137Q、230T和514N在某些条件下提高了效率。
