CRYSTALLIZATION OF YEAST OROTIDINE 5'-MONOPHOSPHATE DECARBOXYLASE COMPLEXED WITH 1-(5'-PHOSPHO-BETA-D-RIBOFURANOSYL) BARBITURIC-ACID

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (K(i) = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 mu-M dimer) plus 450 mu-M BMP by microdialysis at 4-degrees-C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 mu-M BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 angstrom, c = 385 angstrom. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase:BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 angstrom 3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 angstrom resolution over a period of several weeks at room temperature and are isomorphous.