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Crystallization of yeast orotidine 5′‐monophosphate decarboxylase complexed with 1‐(5′‐phospho‐β‐ D ‐ribofuranosyl) barbituric acid
Author(s) -
Bell Juliette B.,
Jones Mary Ellen,
Carter Charles W.
Publication year - 1991
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340090208
Subject(s) - yeast , barbituric acid , crystallization , chemistry , biochemistry , glutamate decarboxylase , stereochemistry , enzyme , organic chemistry
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‐β‐ 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 × 10 −12 M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 μM dimer) plus 450 μM BMP by microdialysis at 4°C against a mother liquor which consisted of 0.1 M Na‐PIPES‐acetate (pH 6.4), 37.5 μM 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 R 32 with unit cell dimensions a = b = 115 Å, c = 385 Å. The crystal density is 1.245 g/cm 3 indicating the presence of two ODCase:BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 Å 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 Å resolution over a period of several weeks at room temperature and are isomorphous.