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, Zhi-Cheng Xiao, Zhi-Jie Liu

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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 71.7 kDa, 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 3.7 A resolution. The crystal unit-cell parameters are a = 91.36, b = 172.03, c = 358.86 A, alpha = beta = gamma = 90.00 degrees . 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 2.19 A(3) Da(-1) with 43.83 solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.
Original languageEnglish
Pages (from-to)1206 - 1210
Number of pages5
JournalActa Crystallographica Section F: Structural Biology Communications
Issue number9
Publication statusPublished - 2014

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