Purification, crystallization and preliminary X-ray analysis of glucokinase from Streptomyces griseus in complex with glucose

Abstract

Glucokinase catalyzes the phosphorylation of glucose using ATP to yield glucose 6-phosphate. SgGlkA is a bacterial group III glucokinase from Streptomyces griseus that seems to play a regulatory role in carbon catabolite repression in this organism. SgGlkA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. A crystal of SgGlkA in complex with glucose was obtained using a reservoir solution consisting of 0.9 M sodium/potassium tartrate, 0.2 M NaCl and 0.1 M imidazole pH 8.1 and diffracted X-rays to 1.84 Å resolution. The crystal of SgGlkA in complex with glucose belonged to space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 109.19, c = 141.18 Å. The crystal contained one molecule in the asymmetric unit.

Figure 1

Purification of SgGlkA by anion-exchange chromatography. Peak 1 represents SgGlkA, which eluted at an NaCl concentration of 0.32 M. Inset: SDS–PAGE gel (15%) for peak 1; molecular-weight markers are labelled in kDa. The approximate molecular weight of SgGlkA is 33 kDa.

Figure 2

Crystals of SgGlkA in complex with glucose.

Figure 3

X-ray diffraction image of SgGlkA in complex with glucose. The circle indicates a resolution of 1.84 Å.