Structural Basis for the Reaction Mechanism of UDP-Glucose Pyrophosphorylase

Abstract

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate 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 product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg(2+)-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 subunit. 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 Mg(2+) 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 reaction is catalyzed by an ordered sequential Bi Bi mechanism. 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 UGPases, as well as a platform for the development of novel antibacterial agents.

Structure and Function of Prokaryotic UDP-Glucose Pyrophosphorylase, A Drug Target Candidate

“…With this objective in mind, several research groups provided crystallographic structures of UGPs from different bacterial sources [14][15][16][17].…”

“…UDP-glucose phosphorylase belongs to the superfamily of the NTP:sugar nucleotidyltransferases (nucleoside diphosphate sugar pyrophosphorylases), a related group of proteins that exhibit a similar folding [17] and certain conserved sequence elements, most notably the N-terminal motif GXGTRXLPXT [18], carrying a putative catalytic arginine residue [17,19]. Apart from the mentioned features, it is characteristic of bacterial UGPs the presence of the signature motif VEKP, which carries essential residues involved in interaction with the glucosyl part of its substrates [15,17] (fig.…”

Crystal Structure of Archaeoglobus fulgidus CTP:Inositol-1-Phosphate Cytidylyltransferase, a Key Enzyme for Di- myo -Inositol-Phosphate Synthesis in (Hyper)Thermophiles

“…The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X 4 -P-K. CTP, L-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.Enzymes classified in the nucleoside triphosphate-transferase family (PF00483) typically transfer nucleoside monophosphate (NMP) from nucleoside triphosphates (NTP) to an acceptor phosphoryl group belonging to a small molecule such as phosphocholine, hexose-1-phosphate, or ribitol-5-phosphate (2,16,20). This activity leads to release of pyrophosphate and production of a nucleoside diphospho-acceptor that is subsequently utilized by glycosyltransferases in a myriad of reactions of vital importance for the cellular functions.…”

“…Enzymes classified in the nucleoside triphosphate-transferase family (PF00483) typically transfer nucleoside monophosphate (NMP) from nucleoside triphosphates (NTP) to an acceptor phosphoryl group belonging to a small molecule such as phosphocholine, hexose-1-phosphate, or ribitol-5-phosphate (2,16,20). This activity leads to release of pyrophosphate and production of a nucleoside diphospho-acceptor that is subsequently utilized by glycosyltransferases in a myriad of reactions of vital importance for the cellular functions.…”

Activated Sugar Precursors: Biosynthetic Pathways and Biological Roles of an Important Class of Intermediate Metabolites in Bacteria

“…This enzyme catalyses the reversible formation of UDP-glucose from UTP and glucose-1-phosphate ( Fig. 8; Kim et al, 2010).…”