X-Ray Structure and Molecular Dynamics Study of Uridine Phosphorylase from Vibrio cholerae in Complex with 2,2'-Anhydrouridine

Crystallization in Microgravity and the Atomic-Resolution Structure of Uridine Phosphorylase from Vibrio cholerae

“…In the subunits A, C, and F, the side chain of Arg47 involved in the phosphate-binding site adopts alternative conformations. The alternative conformations of this residue were observed earlier [6,10,11] in all structures, in which the phosphate-binding site does not contain coordinated orthophosphate or sulfate. As shown previously, the mobility of the side chain of arginine involved in the phosphate-binding site plays an important role in the binding of the orthophosphate anion and its arrangement with respect to the nucleoside molecule [10].…”

“…However, there is a significant difference. Thus, in all the active sites of the structure under consideration, like in 6EYP, Thr93 that forms the flexible wall of the active site [6,10,11] is not hydrogen-bonded to the alcohol molecule and its side chain is at a distance of longer than 4-5 Å from the ligands. Unlike glycerol, the substrates and inhibitors are linked to the side chain of Thr93 through the O4'hydroxy group, the position of which corresponds to the O2-hydroxy group of glycerol.…”

“…Alternative conformations of long secondary-structure elements (β-stands and loops) of the protein tertiary structure in the absence of specific bound ligands were analyzed. Previously, alternative conformations in the atomic-resolution UPh structures were observed only in complexes of UPh with functionally important ligands bound at partial occupancy [6,10,11].…”

“…Currently, great attention is given to the evaluation of the enzyme specificity of UPh and the development of reversible competitive inhibitors, the decrease in the concentration of which leads to the restoration of UPh activity. The structures of the protein from different organisms and its complexes with various ligands were determined and characterized at a resolution of up to 1.2 Å [4][5][6][7][8][9][10][11][12][13].…”

“…This issue can be addressed by determining the crystal structures of the protein and its complexes with high accuracy by means of X-ray crystal-lography. According to our experience, an analysis of the structures determined at atomic and subatomic resolution makes it possible not only to reveal the structural features of the protein and its complexes but also to elucidate the mechanism of action of the enzyme as a biological machine relying on experimental data by analyzing alternative conformations, anisotropic displacement parameters, and active-site protonation states [6,10,11].…”