Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase.
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.
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Chemistry 405C, Baltimore, MD 21250, USA.
Stephen
S
Quirk
Katherine L
KL
Seley-Radtke
D003460:Crystallization; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009713:Nucleotidyltransferases; C026916:fucose-1-phosphate guanylyltransferase