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 (Ki = 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 microM dimer) plus 450 microM 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 microM 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 A, c = 385 A. 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 A3/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 A resolution over a period of several weeks at room temperature and are isomorphous.
Bell
J B
JB
Department of Biochemistry and Biophysics, School of Medicine University of North Carolina, Chapel Hill 27599.
Jones
M E
ME
Carter
C W
CW
D000645:Ammonium Sulfate / Q000494:pharmacology; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction; D015003:Yeasts / Q000201:enzymology*
C011569:6-azauridine-5'-monophosphate; C028372:1-(5'-phospho-beta-D-ribofuranosyl)barbituric acid; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase; D000645:Ammonium Sulfate