细胞表面糖蛋白的岩藻糖基化在许多生理过程中起着至关重要的作用，如Lewis血型抗原的形成、选择素介导的白细胞粘附、Notch信号通路的传导、宿主与微生物间的相互作用以及可作为某些癌症的生物标志物等。GDP-岩藻糖是岩藻糖基化聚糖合成中重要的L-岩藻糖供体。在哺乳细胞中GDP-岩藻糖的合成途径有两种，分别为从头合成途径（de novo pathway）和补救合成途径（salvage pathway）。由于GDP-岩藻糖的重要性及其造价昂贵，体外合成GDP-岩藻糖亟需一种易于操作、高效稳定和成本合理的化学酶合成方法。L-岩藻糖激酶/GDP-岩藻糖焦磷酸化酶（FKP）是一种双功能酶，它可以通过GDP-岩藻糖补救合成途径独立完成从L-岩藻糖合成GDP-岩藻糖的过程。首先，位于FKP蛋白C端的L-岩藻糖激酶在ATP的参与下可与底物L-岩藻糖反应生成L-岩藻糖-1-磷酸；接着，位于FKP蛋白N端的GDP-岩藻糖焦磷酸化酶在GTP的存在下可与中间产物L-岩藻糖-1-磷酸反应生成GDP-岩藻糖。目前FKP蛋白已成为GDP-岩藻糖和岩藻糖基化聚糖生物合成过程中的常用工具酶。在本研究中，我们首次解析了来自脆弱拟杆菌（Bacteroides fragilis 9343）的全长FKP蛋白4.2 ?的冷冻电镜结构，其C端L-岩藻糖激酶结构域分辨率为3.9 ?。在结构中，我们发现细菌FKP蛋白处于四聚体形态，其N端GDP-岩藻糖焦磷酸化酶结构域具有Rossmann折叠和left-handed β-helix折叠特征，C端L-岩藻糖激酶结构域具有典型的GHMP糖激酶折叠特征，linker结构域由5个α螺旋组成。综合序列比对结果和三维结构分析，我们通过氨基酸突变实验分别找到了数个与GTP、ATP和底物L-岩藻糖结合密切相关的氨基酸。另外，通过破坏细菌FKP蛋白分子间作用界面我们获得了单体形态的FKP蛋白，通过酶活测定实验发现单体FKP蛋白的L-岩藻糖酶活性是野生型细菌FKP蛋白的近9倍，GDP-岩藻糖焦磷酸化酶活性与野生型类似。这将极大地促进GDP-岩藻糖的体外合成。为了进一步提高细菌FKP蛋白的分辨率，我们还成功表达和纯化了FKP的N端和C端的截断体蛋白，并获得了细菌FKP蛋白C端截断体的晶体。综上所述，我们解析了全长细菌FKP蛋白4.2 ?的冷冻电镜结构，并通过生化实验揭示了其关键的底物结合位点，为理解FKP蛋白的催化机制提供了帮助。
Fucosylated polysaccharides play crucial roles in several biological processes, including cell-cell recognition, selectin-mediated leukocyte-endothelial adhesion, atherosclerosis development, host-microbe interaction and Lewis blood group antigens formation. GDP-fucose is an essential component for fucosylated polysaccharides synthesis. However, the chemical synthesis of GDP-fucose is a tedious and time-consuming process. L-fucokinase/GDP-fucose pyrophosphorylase (FKP) is a bi-functional enzyme which can produce GDP-fucose from L-fucose in a step-by-step manner. Study of FKP will provide valuable insights into the biosynthesis of GDP-fucose and fucosylated polysaccharides. In the present study, we reported a cryo-EM structure of the homotetrameric FKP from Bacteroides fragilis 9343 at an overall resolution of 4.2 ?, with the C-terminal fucokinase domain at 3.9 ?. The N-terminal domain, which has the GDP-fucose pyrophosphorylase activity, contains a Rossmann fold and a left-handed β-helix fold. And the C-terminal domain, which has the L-fucokinase activity, adopts a typical GHMP sugar kinase fold. Mutagenesis and biochemical studies based on sequence alignment results and structural information revealed the nucleotides and fucose binding sites in FKP. Additionally, disrupting one of intermolecular interfaces through mutagenesis can turn the tetrameric FKP into a monomer. The monomeric FKP has a 9-fold higher L-fucokinase activity comparing with the wild type, meanwhile has a similar GDP-fucose pyrophosphorylase activity. This will greatly facilitate the GDP-fucose synthesis in vitro. Moreover, we also have expressed and purified the N- and C-terminal truncated protein of FKP successfully and got the crystal of FKP C-terminal truncation. This will be rather helpful for the resolution improvement of FKP in the future.In conclusion, we resolve the first full-length cryo-EM structure of FKP at 4.2 ? and matagenesis and biochemical studies reveal the substrate-binding sites in FKP. This study provide a better understanding of the catalytic mechanism of FKP.
