Polymorphism of microcrystalline urate oxidase from Aspergillus flavus.
Different polymorphs of rasburicase, a recombinant urate oxidase enzyme (Uox) from Aspergillus flavus, were obtained as a series of polycrystalline precipitates. Different crystallization protocols were followed in which the salt type, pH and polyethylene glycol 8000 (PEG 8000) concentration were varied. The related crystalline phases were characterized by means of high-resolution synchrotron X-ray powder diffraction. In all cases, Uox complexed with the inhibitor 8-azaxanthine (AZA) was not altered from its robust orthorhombic I222 phase by variation of any of the factors listed above. However, in the absence of AZA during crystallization ligand-free Uox was significantly affected by the type of salt, resulting in different crystal forms for the four salts tested: sodium chloride, potassium chloride, ammonium chloride and ammonium sulfate. Remarkable alterations of some of these phases were observed upon gradual increase of the exposure time of the sample to the synchrotron beam in addition to variation of the PEG 8000 concentration. When Uox was crystallized in Tris buffer or pure water in the absence of salt, a distinct polymorph of orthorhombic symmetry (P2(1)2(1)2) was obtained that was associated with significantly altered lattice dimensions in comparison to a previously reported isosymmetrical structure. The latter form of Uox exhibits enhanced stability to variation of pH and PEG 8000 concentration accompanied by minor modifications of the unit-cell dimensions in the ranges under study. Accurate lattice parameters were extracted for all crystalline phases. This study reveals the rich phase diagram of Uox, a protein of high pharmaceutical importance, which is associated with an enhanced degree of polymorphism. The outcome of our analysis verifies previously reported results as well as demonstrating polymorphs that have altered unit-cell dimensions with respect to known structural models.
Collings
Ines
I
European Synchrotron Radiation Facility, BP-220, F-38043 Grenoble CEDEX 9, France.
Watier
Yves
Y
Giffard
Marion
M
Dagogo
Sotonye
S
Kahn
Richard
R
Bonneté
Francoise
F
Wright
Jonathan P
JP
Fitch
Andrew N
AN
Margiolaki
Irene
I
D001231:Aspergillus flavus / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D044367:Phase Transition; D014503:Urate Oxidase / Q000737:chemistry*
D014503:Urate Oxidase