title: Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase from Bacteroides fragilis.
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 71.7 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 Å resolution. The crystal unit-cell parameters are a = 91.36, b = 172.03, c = 358.86 Å, 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 2.19 Å(3) Da(-1) with 43.83% solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne 3800, Australia.
authors.forename: Chongyun
authors.initials: C
authors.lastname: Cheng
authors.affiliation: Department of General Surgery, Tianjin First Center Hospital, Tianjin 300192, People's Republic of China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Gu
authors.affiliation: National Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Su
authors.affiliation: Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
authors.affiliation: National Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yin
authors.affiliation: National Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
authors.forename: Wenguang
authors.initials: W
authors.lastname: Liang
authors.affiliation: National Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne 3800, Australia.
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.affiliation: Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne 3800, Australia.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: National Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
mesh_terms: D001441:Bacteroides fragilis / Q000201:enzymology*; D002850:Chromatography, Gel; D020285:Cryoelectron Microscopy; D003460:Crystallization; D018360:Crystallography, X-Ray; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism
chemical_list: D017853:Phosphotransferases (Alcohol Group Acceptor); C020153:fucokinase; D009713:Nucleotidyltransferases; C020842:guanylyltransferase
keywords: Bacteroides fragilis; FKP; cryo-EM