Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase

• Published in: Molecules and cells Vol. 29; no. 4; pp. 397 - 405
• Main Authors: Kim, H., Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea, Choi, J.K., Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea, Kim, Truc, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea, Lokanath, Neratur K., University of Mysore, Mysore, India, Ha, S.C., Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea, Suh, S.W., Seoul National University, Seoul, Republic of Korea, Hwang, H.Y., Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea, Kim, K.K., Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Springer Korean Society for Molecular and Cellular Biology 01.04.2010; 한국분자세포생물학회
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biomedical and Life Sciences; Biomedicine; Biotechnology; Calorimetry - methods; Catalysis; Catalytic Domain - genetics; Cell Biology; crystal structure; Crystallography, X-Ray; GLUCOSA; GLUCOSE; glucose-1-phosphate uridylyltransferase; Glucosephosphates - chemistry; Glucosephosphates - metabolism; Helicobacter pylori - enzymology; isothermal titration calorimetry; Kinetics; Life Sciences; Magnesium - chemistry; Magnesium - metabolism; Models, Chemical; Models, Molecular; Molecular Structure; Mutation; Protein Conformation; Protein Structure, Tertiary; Quantitative Structure-Activity Relationship; reaction mechanism; Substrate Specificity; UDP-glucose pyrophosphorylase; Uridine Diphosphate Glucose - chemistry; Uridine Diphosphate Glucose - metabolism; UTP-Glucose-1-Phosphate Uridylyltransferase - chemistry; UTP-Glucose-1-Phosphate Uridylyltransferase - genetics; UTP-Glucose-1-Phosphate Uridylyltransferase - metabolism; 생물학; crystal structure; glucose-1-phosphate uridylyltransferase; UDP-glucose pyrophosphorylase; isothermal titration calorimetry; reaction mechanism
• ISSN: 1016-8478; 0219-1032
• DOI: 10.1007/s10059-010-0047-6

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/Mg²+-bound forms at 2.9 latin capital letter A with ring above and 2.3 latin capital letter A with ring above 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 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 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.