Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/l-fucose-1-P-guanylyltransferase from Bacteroides fragilis

Chongyun Cheng, Jianhua Gu, Jing Su, Wei Ding, Jie Yin, Wenguang Liang, Xiaoxia Yu, Jun Ma, Peng George Wang, Zhicheng Xiao, Zhi Jie Liu

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

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 a salvage pathway. The molecular weights of full-length FKP (F-FKP) and C-terminally truncated FKP (C-FKP, residues 300-949) are 105.7 and 71.7kDa, respectively. In this study, both recombinant F-FKP and C-FKP were expressed and purified. Size-exclusion chromatography experiments and analytical ultracentrifugation results showed that both F-FKP and C-FKP are trimers. Native F-FKP protein was crystallized by the vapour-diffusion method and the crystals belonged to space group P212121 and diffracted synchrotron X-rays to 3.7Å resolution. The crystal unit-cell parameters are a = 91.36, b = 172.03, c = 358.86Å, α = β = γ = 90.00°. The three-dimensional features of the F-FKP molecule were observed by cryo-EM (cryo-electron microscopy). The preliminary cryo-EM experiments showed the F-FKP molecules as two parallel disc-shaped objects stacking together. Combining all results together, it is assumed that there are six FKP molecules in one asymmetric unit, which corresponds to a calculated Matthews coefficient of 2.19Å3Da-1 with 43.83% solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.

Original languageEnglish (US)
Pages (from-to)1206-1210
Number of pages5
JournalActa Crystallographica Section:F Structural Biology Communications
Volume70
DOIs
StatePublished - Sep 1 2014
Externally publishedYes

Keywords

  • Bacteroides fragilis
  • cryo-EM
  • FKP

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Genetics
  • Condensed Matter Physics

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