Download Combined X-ray and neutron crystallography studies of urate oxidase

Combined X-ray and neutron crystallographic studies of urate
oxidase
Monika Budayova-Spano
UJF-EMBL-CNRS, Grenoble, France
Urate oxidase is an enzyme which catalyses the oxidation of uric acid to allantoin. It is
produced and commercialized by Sanofi-Synthélabo to be used as a protein drug to reduce
toxic uric acid accumulation and to resolve the hyperuricemic disorders occurring during
chemotherapy. Several X-ray structures have been solved for Uox in complex with various
uric acid analogues [1-3] and a putative mechanism for the oxidation of uric acid has been
proposed. However, the precise ionization state of the substrate during the reaction is not yet
definitively established. Co-crystallization with the substrate as well as obtaining the large
high-quality crystals that are required to compensate for the weak flux of available neutron
sources [4] become a mandatory stage to line out the enzyme active site. A novel method
and apparatus [5] that found application in the growth of large high-quality crystals of
hydrogenated urate oxidase in complex with its substrate analogues as well as with its
natural substrate will be described. The high-resolution X-ray and neutron diffraction data
were collected from these crystals on the id23 instrument at the ESRF and on the LADI III
instrument at the ILL. The contribution of the combined X ray & neutron crystallographic
studies would be discussed in term of the implications on the enzymatic mechanism of this
therapeutically important enzyme. This work represents the first step in providing evidence
on the protonation state of the inhibitor and residues within the active site. [1] Colloc'h, N., El
Hajji, M., Bachet, B., Lhermite, G., Schiltz, M., Prangé, T., Castro B. and Mornon, J. P.
(1997) Nature Struct. Biol. 4, 947-952. [2] Retailleau, P. Colloc'h, N., Vivarès, D., Bonneté,
F., Castro, B., El Hajji, M.,.Mornon, J.P, Monard, G. and Prangé, T. (2004) Acta Cryst. D60,
453-462. [3] Retailleau, P., Colloc'h, N., Vivarès, D., Bonneté, F., Castro, B., El Hajji and
Prangé, T. (2005) Acta Cryst. D61, 218-229. [4] Myles, D.A.A., Bon, C., Langan, P., Cipriani,
F., Castagna, J.C., Lehmann, M.S., Wilkinson, C. (1998). Physica B, 241-243, 1122-1130. [5]
Budayova-Spano, M., Dauvergne, F., Audiffren, M., Bactivelane, T., Cusack, S. (2007) Acta
Cryst. D63, 339-347. Combined X-ray and neutron crystallographic studies of urate oxidase
Oral abstract Urate oxidase is an enzyme which catalyses the oxidation of uric acid to
allantoin. It is produced and commercialized by Sanofi-Synthélabo to be used as a protein
drug to reduce toxic uric acid accumulation and to resolve the hyperuricemic disorders
occurring during chemotherapy. Several X-ray structures have been solved for Uox in
complex with various uric acid analogues [1-3] and a putative mechanism for the oxidation of
uric acid has been proposed. However, the precise ionization state of the substrate during
the reaction is not yet definitively established. Co-crystallization with the substrate as well as
obtaining the large high-quality crystals that are required to compensate for the weak flux of
available neutron sources [4] become a mandatory stage to line out the enzyme active site. A
novel method and apparatus [5] that found application in the growth of large high-quality
crystals of hydrogenated urate oxidase in complex with its substrate analogues as well as
with its natural substrate will be described. The high-resolution X-ray and neutron diffraction
data were collected from these crystals on the id23 instrument at the ESRF and on the LADI
III instrument at the ILL. The contribution of the combined X ray & neutron crystallographic
studies would be discussed in term of the implications on the enzymatic mechanism of this
therapeutically important enzyme. This work represents the first step in providing evidence
on the protonation state of the inhibitor and residues within the active site. [1] Colloc'h, N., El
Hajji, M., Bachet, B., Lhermite, G., Schiltz, M., Prangé, T., Castro B. and Mornon, J. P.
(1997) Nature Struct. Biol. 4, 947-952. [2] Retailleau, P. Colloc'h, N., Vivarès, D., Bonneté,
F., Castro, B., El Hajji, M.,.Mornon, J.P, Monard, G. and Prangé, T. (2004) Acta Cryst. D60,
453-462. [3] Retailleau, P., Colloc'h, N., Vivarès, D., Bonneté, F., Castro, B., El Hajji and
Prangé, T. (2005) Acta Cryst. D61, 218-229. [4] Myles, D.A.A., Bon, C., Langan, P., Cipriani,
F., Castagna, J.C., Lehmann, M.S., Wilkinson, C. (1998). Physica B, 241-243, 1122-1130. [5]
Budayova-Spano, M., Dauvergne, F., Audiffren, M., Bactivelane, T., Cusack, S. (2007) Acta
Cryst. D63, 339-347.