Crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from Thermus thermophilus

Abstract

Glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. The open reading frame TTHA0299 of the extreme thermophile Thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. In this study, the glucokinase/hexokinase from T. thermophilus was purified and crystallized using polyethylene glycol 8000 as a precipitant. Diffraction data were collected and processed to 2.02 Å resolution. The crystal belonged to space group P2(1), with unit-cell parameters a = 70.93, b = 138.14, c = 75.16 Å, β = 95.41°.

Figure 1

Sequence alignment of TthGK/HK and homologous proteins. The white and black bars indicate consensus sequences 1 and 2, respectively, of TthGK/HK. Multiple alignments were performed by ClustalW (Higgins et al., 1996 ▶). Tth, TthGK/HK (AAS82030); Tca, HK from Thermus caldophilus (AAV66920); Tma, GK from Thermotoga maritima (AAD36537); Ape, HK from Aeropyrum pernix (BAA81102); Tte, HK from Thermoproteus tenax (CAD52839); Eco, GK from E. coli (ACI80495); Pfu, GK from Pyrococcus furiosus (AAL80436); Pho; GK from P. horikoshii (BAA29678); Tli, GK from Thermococcus litoralis (Q7M537). GenBank/Swiss-Prot accession numbers are given in parentheses.

Figure 2

Crystals of TthGK/HK.

Figure 3

Diffraction image of TthGK/HK. Circles corresponding to 2.0 and 2.5 Å resolution are shown.

Figure 4

Self-rotation function calculated in the (a) χ = 180° and (b) χ = 90° sections.

Figure 5

Gel-filtration chromatogram of TthGK/HK and calibration curve (inset). The standard proteins are (1) catalase, (2) aldolase, (3) albumin and (4) ovalbumin. The estimated molecular mass of TthGK/HK is 131.9 kDa, while the calculated monomer mass is 31 479 Da.