UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-pho-sphate to UDP-glucose and pyrophosphate and vice versa.
Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their prod-
uct, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UG-Pase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg²⁺-bound forms at 2.9 A and 2.3 A resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each sub-unit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules
with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg²⁺ ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reac-
tion is catalyzed by an ordered sequential Bi Bi mecha-nism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UG-Pases, as well as a platform for the development of novel antibacterial agents.

Keywords: crystal structure, glucose-1-phosphate uridylyltransferase, isothermal titration calorimetry, reaction mechanism, UDP-glucose pyrophospho-rylase