Cloning, expression, purification, crystallization and preliminary X-ray analysis of a fructokinase from Vibrio cholerae O395

Abstract

Fructokinase (FK), one of the crucial enzymes for sugar metabolism in bacterial systems, catalyses the unidirectional phosphorylation reaction from fructose to fructose 6-phosphate, thereby allowing parallel entry of fructose into glycolysis beside glucose. The cscK gene from Vibrio cholerae O395 coding for the enzyme FK has been cloned, overexpressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Crystals of V. cholerae FK (Vc-FK) and its cocrystal with fructose, adenosine diphosphate (ADP) and Mg2+ were grown in the presence of polyethylene glycol 6000 and diffracted to 2.45 and 1.75 Å resolution, respectively. Analysis of the diffraction data showed that both crystal forms have symmetry consistent with space group P2(1)2(1)2, but with different unit-cell parameters. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient for the apo Vc-FK crystals was estimated to be 2.4 Å3 Da(-1), which corresponds to a solvent content of 48%. The corresponding values for the ADP- and sugar-bound Vc-FK crystals were 2.1 Å3 Da(-1) and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.

Figure 1

The homogeneity of the purified Vc-FK protein was checked by 12% SDS–PAGE. Lane 1, Vc-FK; lane 2, molecular-mass markers (labelled in kDa).

Figure 2

(a) Crystals of Vc-FK in the native form grown in the presence of 5% PEG 6000 pH 6 at 293 K. The maximum dimensions of the crystals were 0.4 × 0.2 × 0.3 mm. (b) Cocrystals of Vc-FK with fructose, ADP and MgCl₂ of 0.4 × 0.3 × 0.2 mm in size appeared at 293 K when 5% PEG 6000 pH 6.0 was used as a precipitant.

Figure 3

(a) X-ray diffraction image of an apo Vc-FK crystal; the region in the black square is enlarged to show the diffraction at the edge, which corresponds to a resolution of 2.45 Å (black arrow). (b) X-ray diffraction image of a Vc-FK–ADP–fructose cocrystal; the region in the black square is enlarged to show the diffraction at the edge, which corresponds to a resolution of 1.75 Å (black arrow).