Crystallization and preliminary X-ray study of Vibrio cholerae uridine phosphorylase in complex with 6-methyluracil

Abstract

Uridine phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-position, such as 6-methyluracil (6-MU). In order to explain this fact, it is essential to investigate the three-dimensional structure of the complex of 6-MU with uridine phosphorylase. 6-MU is a pharmaceutical agent that improves tissue nutrition and enhances cell regeneration by normalization of nucleotide exchange in humans. 6-MU is used for the treatment of diseases of the gastrointestinal tract, including infectious diseases. Here, procedures to obtain the uridine phosphorylase from the pathogenic bacterium Vibrio cholerae (VchUPh), purification of this enzyme, crystallization of the complex of VchUPh with 6-MU, and X-ray data collection and preliminary X-ray analysis of the VchUPh-6-MU complex at atomic resolution are reported.

Keywords: 6-methyluracil; Vibrio cholerae; bacterial uridine phosphorylase.

Figure 1

Scheme showing the reaction catalyzed by uridine phosphorylase: (a) reversible interconversion of the native substrate uridine to uracil and (b) irreversible conversion of the substrate analogue 6-methyluridine to 6-methyluracil. Conventional nomenclature: P, phosphorolysis reaction; S, nucleoside-synthesis reaction.

Figure 2

Crystals of uridine phosphorylase from V. cholerae in complex with 6-MU.

Figure 3

Nucleoside-binding site of VchUPh in complex with 6-MU (prepared using PyMOL v.0.99; DeLano, 2002 ▶).