Poster Presentation 2014 International Biophysics Congress

Crystallization, preliminary X-Ray crystallographic and Cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase from B. Fragilis (#346)

Chongyun Cheng 1 2 , Jianhua Gu 3 , Jing Su 2 , Wei Ding 2 , Jie Yin 2 , Wenguang Liang 2 , Xiaoxia Yu 2 , Jun Ma 1 , Peng Wang 4 , Zhicheng Xiao 1 , Zhijie Liu 2
  1. Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia
  2. National Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
  3. Department of General Surgery, Tianjin First Center Hospital, Tianjin, China
  4. Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA
Fucokinase/L-fucose-1-P-guanylyltransferase (FKP) is a bifunctional enzyme which converts L-fucose to Fuc-1-P and thence to GDP-L-fucose through the salvage pathway. The molecular weights of the full length FKP (F-FKP) and the C terminal truncated FKP (C-FKP, residues 300-949 amino acid) are 105.7 kDa and 71.7 kDa, respectively. In this study, both recombinant F-FKP and C-FKP were expressed and purified. Size exclusion experiments showed both F-FKP and C-FKP have two different oligomeric states and the lower oligomerics were predominant. Analytical ultracentrifugation results showed the lower oligomerics of F-FKP and C-FKP are trimers. F-FKP native protein was crystallized by vapour diffusion and its three dimensional feature was observed by Cryo-EM. The crystals belong to the space group P212121 and diffracted synchrotron X-rays to 3.7Å resolution. Crystal unit cell parameters are a=91.36Å, b=172.03Å, c=358.86Å, α=90.00º, β=90.00º, γ=90.00º, respectively. Considering the calculated Matthews coefficient of 2.19 with 43.83% solvent content, six molecules in one asymmetric unit is proposed. The Cryo-EM image showed F-FKP molecules is a-two-parallel disk-shaped objects stacking together, indicating two trimers are likely present in one asymmetric unit. These preliminary crystallographic and oligomeric analyse provide basic structure information of FKP.