Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states inAspergillus flavusurate oxidase

Abstract

Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05 -1.20 Å ) and neutron diffraction data (1.9 -2.5 Å ) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H 2 O and D 2 O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis.Keywords: urate oxidase; neutron and X-ray crystallography; crystal growth; phase diagram; H -D exchange; protonation states INTRODUCTIONUrate oxidase (uricase or Uox, EC 1.7.3.3) is an enzyme involved in purine metabolism. It catalyses the oxidation of uric acid to 5-hydroxyisourate, a metastable intermediate, which is further degraded to allantoin. Humans and other primates lack a functional Uox, and therefore the final product of purine metabolism is uric acid. It has been speculated that this confers an evolutionary advantage to long-lived animals like primates owing to the anti-oxidant properties of urate, but the downside is that in certain conditions high urate levels (hyperuricaemia) may lead to crystallization of uric acid, as in gout.Uox from Aspergillus flavus has been commercialized by Sanofi-Aventis as a protein drug to treat hyperuraemic conditions such as the tumour lysis syndrome, but the catalytic mechanism of Uox is still poorly understood. Several X-ray structures have been solved for Uox in the complex with various uric acid analogues (Colloc'h et al. 1997(Colloc'h et al. , 2007Retailleau et al. 2004Retailleau et al. , 2005Gabison et al. 2006Gabison et al. , 2008, and a putative mechanism for the oxidation of uric acid has been proposed. However, many of the key questions are related to the protonation state of the substrate during the reaction. Uric acid has four potentially acidic protons; this gives rise to four different monoanions and six dianions. To be able to describe the mechanism, it is necessary to know which of the anions is the real substrate and how the hydrogen bonding netw...

Structure, function and applications of carbonic anhydrase isozymes

“…24,58 The Val121 is present at the mouth of the hydrophobic pocket in the active site of the enzyme, was mutated to Gly, Ala, Ser, Leu, Ile, Lys, and Arg. [59][60][61][62][63][64][65][66][67] It was observed that pnitrophenyl esterase activity and the CO 2 hydrase activity of these CAII variants are directly related to the hydrophobicity of these residues. Therefore, it was recommended that hydrophobic character of such residues is essentially important for catalysis.…”

“…Therefore, it was recommended that hydrophobic character of such residues is essentially important for catalysis. [59][60][61][62][63][64][65][66][67] The active site of CA is present in the deep, and one side is lined by hydrophobic residues, while hydrophilic residues like Thr199 and Glu106 are resting on the other side. At deep cleft Zn 2+ ion synchronized tetrahedrally with imidazoles of three conserved histidines (His94, His96 and His119) and a water molecule as a fourth solvent ligand 42 .…”

A historical perspective on protein crystallization from 1840 to the present day

“…A [316]. Another paradigm change occurred with the advent of sophisticated X-ray optics, ultrasensitive detectors and microbeams at new-generation synchrotron sources [288].…”

“…Large crystals are required for neutron crystallography and, for that purpose, the crystal growth technique based on temperature variations is particularly appropriate . It has been applied to grow high‐quality large crystals of several proteins of interest, which, in the case of A. flavus urate oxidase, yielded neutron diffraction data in the range 1.9–2.5 Å .…”

Macromolecular Crystallization Controlled by Colloidal Interactions: The Case of Urate Oxidase

“…One possible mechanism for the oxidation of uric acid could be revealed by the precise protonation state of the substrate during the reaction. Neutron crystallography can provide such information and make possible direct determination of the protonation states of the active site residues and substrate analogues (Oksanen et al, 2009). For this purpose, it is necessary to grow large, well ordered, deuterated crystals; here too, therefore, a good knowledge of the phase diagram is required.…”