Ma Wei, Yuan Shiyu, Wang Zixian, Niu Kangle, Li Fengyi, Liu Lulu, Han Lijuan, Fang Xu
State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China.
Rongcheng Huihai Chuangda Biotechnology Co., Ltd., Weihai 264300, China.
Eng Microbiol. 2024 May 22;4(4):100151. doi: 10.1016/j.engmic.2024.100151. eCollection 2024 Dec.
This research identified four amino acid residues (Leu174, Asn297, Tyr301, and Gln291) that contribute to substrate recognition by the high-affinity glucose transporter Xltr1p from . Potential hotspots affecting substrate specificity were selected through homology modeling, evolutionary conservation analyses, and substrate-docking modeling of Xltr1p. Variants carrying mutations at these hotspots were subsequently obtained via in silico screening. Replacement of Leu174 or Asn297 in Xltr1p with alanine resulted in loss of hexose transport activity, indicating that Leu174 and Asn297 play essential roles in hexose transport. The Y301W variant exhibited accelerated mannose transport, but lost galactose transport capacity, and mutation of Gln291 to alanine greatly accelerated mannose transport. These results suggest that amino acids located in transmembrane α-helix 7 (Asn297, Tyr301, and Gln291) play critical roles in substrate recognition by the hexose transporter Xltr1p. Our results will help expand the potential applications of this transporter and provide insights into the mechanisms underlying its function and specificity.
本研究鉴定出四个氨基酸残基（Leu174、Asn297、Tyr301和Gln291），它们有助于来自[具体来源未给出]的高亲和力葡萄糖转运蛋白Xltr1p识别底物。通过同源建模、进化保守性分析和Xltr1p的底物对接建模，选择了影响底物特异性的潜在热点。随后通过计算机筛选获得了在这些热点处携带突变的变体。将Xltr1p中的Leu174或Asn297替换为丙氨酸导致己糖转运活性丧失，表明Leu174和Asn297在己糖转运中起关键作用。Y301W变体表现出加速的甘露糖转运，但失去了半乳糖转运能力，并且将Gln291突变为丙氨酸极大地加速了甘露糖转运。这些结果表明，位于跨膜α螺旋7中的氨基酸（Asn297、Tyr301和Gln291）在己糖转运蛋白Xltr1p识别底物中起关键作用。我们的结果将有助于扩展该转运蛋白的潜在应用，并为其功能和特异性的潜在机制提供见解。
