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Mol. Cells 2010; 29(4): 397-405

Published online March 15, 2010

https://doi.org/10.1007/s10059-010-0047-6

© The Korean Society for Molecular and Cellular Biology

Structural Basis for the Reaction Mechanism of UDP-Glucose Pyrophosphorylase

Hun Kim, Jongkeun Choi, Truc Kim, Neratur K. Lokanath1, Sung Chul Ha, Se Won Suh2,
Hye-Yeon Hwang, and Kyeong Kyu Kim*

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea, 1Department of Studies in Physics, University of Mysore, Manasagangotri, Mysore 570 006, India, 2Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea

Correspondence to : *Correspondence: kkim@med.skku.ac.kr

Received: September 18, 2009; Revised: December 18, 2009; Accepted: December 30, 2009

Abstract

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

Article

Research Article

Mol. Cells 2010; 29(4): 397-405

Published online March 15, 2010 https://doi.org/10.1007/s10059-010-0047-6

Copyright © The Korean Society for Molecular and Cellular Biology.

Structural Basis for the Reaction Mechanism of UDP-Glucose Pyrophosphorylase

Hun Kim, Jongkeun Choi, Truc Kim, Neratur K. Lokanath1, Sung Chul Ha, Se Won Suh2,
Hye-Yeon Hwang, and Kyeong Kyu Kim*

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea, 1Department of Studies in Physics, University of Mysore, Manasagangotri, Mysore 570 006, India, 2Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea

Correspondence to:*Correspondence: kkim@med.skku.ac.kr

Received: September 18, 2009; Revised: December 18, 2009; Accepted: December 30, 2009

Abstract

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

Mol. Cells
Jul 31, 2022 Vol.45 No.7, pp. 435~512
COVER PICTURE
Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into mesodermal lineages like adipogenic, osteogenic, and chondrogenic. Alcian blue-positive extracellular matrix secreted by chondrocytes in the lacuna confirmed the chondrogenic differentiation of MSCs (Bashyal et al., pp. 479-494).

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