Structural And Biochemical Investigation Of L-Fucokinase/GDP-Fucose Pyrophosphorylase(FKP)

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.