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 Citations (Scopus)

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

Fingerprint

fucokinase
Cryoelectron Microscopy
Bacteroides fragilis
Fucose
Crystallization
Electron microscopy
enzymes
electron microscopy
X-Rays
crystallization
X rays
Enzymes
molecules
x rays
chromatography
trimers
exclusion
crystals
molecular weight
synchrotrons

Keywords

  • Bacteroides fragilis
  • cryo-EM
  • FKP

ASJC Scopus subject areas

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

Cite this

Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/l-fucose-1-P-guanylyltransferase from Bacteroides fragilis. / Cheng, Chongyun; Gu, Jianhua; Su, Jing; Ding, Wei; Yin, Jie; Liang, Wenguang; Yu, Xiaoxia; Ma, Jun; Wang, Peng George; Xiao, Zhicheng; Liu, Zhi Jie.

In: Acta Crystallographica Section:F Structural Biology Communications, Vol. 70, 01.09.2014, p. 1206-1210.

Research output: Contribution to journalArticle

Cheng, Chongyun ; Gu, Jianhua ; Su, Jing ; Ding, Wei ; Yin, Jie ; Liang, Wenguang ; Yu, Xiaoxia ; Ma, Jun ; Wang, Peng George ; Xiao, Zhicheng ; Liu, Zhi Jie. / Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/l-fucose-1-P-guanylyltransferase from Bacteroides fragilis. In: Acta Crystallographica Section:F Structural Biology Communications. 2014 ; Vol. 70. pp. 1206-1210.
@article{378b347d57a24ece9beba4e174b77917,
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 71.7kDa, 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 3.7{\AA} resolution. The crystal unit-cell parameters are a = 91.36, b = 172.03, c = 358.86{\AA}, 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{\AA}3Da-1 with 43.83{\%} solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.",
keywords = "Bacteroides fragilis, cryo-EM, FKP",
author = "Chongyun Cheng and Jianhua Gu and Jing Su and Wei Ding and Jie Yin and Wenguang Liang and Xiaoxia Yu and Jun Ma and Wang, {Peng George} and Zhicheng Xiao and Liu, {Zhi Jie}",
year = "2014",
month = "9",
day = "1",
doi = "10.1107/S2053230X14012722",
language = "English (US)",
volume = "70",
pages = "1206--1210",
journal = "Acta Crystallographica Section F:Structural Biology Communications",
issn = "1744-3091",
publisher = "John Wiley and Sons Ltd",

}

TY - JOUR

T1 - Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/l-fucose-1-P-guanylyltransferase from Bacteroides fragilis

AU - Cheng, Chongyun

AU - Gu, Jianhua

AU - Su, Jing

AU - Ding, Wei

AU - Yin, Jie

AU - Liang, Wenguang

AU - Yu, Xiaoxia

AU - Ma, Jun

AU - Wang, Peng George

AU - Xiao, Zhicheng

AU - Liu, Zhi Jie

PY - 2014/9/1

Y1 - 2014/9/1

N2 - 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.

AB - 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.

KW - Bacteroides fragilis

KW - cryo-EM

KW - FKP

UR - http://www.scopus.com/inward/record.url?scp=84949129927&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84949129927&partnerID=8YFLogxK

U2 - 10.1107/S2053230X14012722

DO - 10.1107/S2053230X14012722

M3 - Article

C2 - 25195892

AN - SCOPUS:84949129927

VL - 70

SP - 1206

EP - 1210

JO - Acta Crystallographica Section F:Structural Biology Communications

JF - Acta Crystallographica Section F:Structural Biology Communications

SN - 1744-3091

ER -