Abstract
The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 angstroms and belong to the orthorhombic space group P2(1)2(1)2(1). There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 angstroms3 Da(-1) and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.