Expression, purification and preliminary crystallographic studies of humanketohexokinase

Ketohexokinase (KHK; E.C. 2.7.1.3) catalyses the (reversible) phosphorylati on of fructose to fructose-1-phosphate. KHK is the first enzyme in a specia lized catabolic pathway metabolizing dietary fructose to the glycolytic int ermediate glyceraldehyde-3-phosphate. Mutations inactivating KHK underlie t he metabolic disorder essential fructosuria. The primary structure of KHK s hows no significant homology to other mammalian hexokinases. It is most sim ilar to prokaryotic ribokinases, but catalyses a distinct phosphorylation r eaction. Recombinant human KHK has been crystallized in the orthorhombic fo rm (space group P2(1)2(1)2 or P2(1)2(1)2(1)). Single crystals of this polym orph suitable for X-ray diffraction have been obtained by vapour diffusion using 2-propanol and MPD as precipitants (pH 7.5). The crystals have unit-c ell parameters a = 93.4, b = 121.5, c = 108.4 Angstrom. Diffraction data we re collected to 4.3 Angstrom resolution. The asymmetric unit contains four protein molecules.