Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii

Galactokinase (EC 2.7.1.6) catalyzes the ATP‐dependent phosphorylation of α‐­d‐­galactose to α‐d‐galactose‐1‐phosphate, in an additional metabolic branch of glycolysis. The apo‐form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with α‐d‐galactose and an ATP analogue. Diffraction data sets were collected to 1.24 Å resolution for the apo form and to 1.7 Å for the ternary complex form using synchrotron radiation. The apo‐form crystals belong to space group C2, with unit‐cell parameters a = 108.08, b = 38.91, c = 81.57 Å, β = 109.8°. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.