Cloning, purification, crystallization and preliminary crystallographic analysis of galactokinase from Pyrococcus furiosus

Galactokinase catalyses the conversion of galactose to galactose-1-phosphate as the first step in the Leloir pathway, a metabolic route that eventually enables the degradation of galactose via the glycolytic pathway. Galactokinases have been isolated from a wide range of prokaryotic and eukaryotic organisms and the enzyme has been identified as a member of the GHMP kinase (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) superfamily. Pyrococcus furiosus galactokinase was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to the space group C222₁, with more than eight subunits in the asymmetric unit and with approximate unit-cell parameters a = 211.7, b = 355.4, c = 165.5 Å, α = β = γ = 90°. The crystals diffract X-rays to 2.9 Å resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the molecular basis of substrate recognition and catalysis of this enzyme, for which no structures are currently available.