ISSN:
0887-3585
Keywords:
BMP
;
ODCase
;
OMP
;
pyrimidine biosynthesis
;
incomplete factorial
;
crystal growth
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
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 (Ki = 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 R32 with unit cell dimensions a = b = 115 Å, c = 385 Å. 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 Å3/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.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.340090208
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