Crystallization of uridine phosphorylase fromShewanella oneidensisMR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis

Abstract

Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP from Shewanella oneidensis MR-1 was performed in the recipient strain Escherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space group P2 1 and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.

Effect of macromolecular mass transport in microgravity protein crystallization

“…The microgravity-grown ribonuclease S single crystals revealed improved diffraction resolution compared to ground-grown crystals of equal size (Asano et al, 1992). In another experiment, crystals of uridine phosphorylase from Shewanella oneidensis MR-1 revealed improved quality for microgravity-grown crystals compared to 1g controls (Safonova et al, 2012). However, some crystallization experiments performed under microgravity conditions showed no significant improvement in crystal quality (Hilgenfeld et al, 1992;Strong et al, 1992).…”

High-synconformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures ofShewanella oneidensisMR-1 uridine phosphorylase in the free form and in complex with uridine

“…The search for crystallization conditions for SoUP in the free form and in complex with uridine has been described in detail previously (Safonova et al, 2012). Crystals were grown by the hanging-drop vapour-diffusion method at room temperature using kits from Hampton Research.…”

Concerted action of two subunits of the functional dimer ofShewanella oneidensisMR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme

“…Previously, we have isolated and characterized UP from the bacterium Shewanella oneidensis MR-1 (SoUP) belonging to the gammaproteobacteria (Mordkovich et al, 2012(Mordkovich et al, , 2013 and have determined the three-dimensional structures of wild-type (WT) SoUP in the free state and in complex with uridine by X-ray diffraction (Safonova et al, 2012(Safonova et al, , 2014. Uridine phosphorylases from different gammaproteobacteria have been characterized by physicochemical methods (Veiko et al, 1994(Veiko et al, , 1998Takehara et al, 2014;Molchan et al, 1998), and the three-dimensional structures of UPs from different sources have been determined, e.g.…”