Abstract
Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel beta-helix (LbetaH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.
Highlights
Uridinediphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9)2 is present in all three kingdoms of life and catalyzes the reaction of UTP ϩ glucose 1-phosphate 3 UDP-glucose ϩ PPi in the presence of Mg2ϩ in vivo
UGPase is involved in the biosynthesis of the lipopolysaccharide core in Escherichia coli, resulting in a reduced adhesion behavior of E. coli galU mutants [4]
UGPase represents a key position in the activation of glucose and galactose, which are major components of Leishmania glycoconjugates
Summary
Protein Expression and Purification—The gene encoding UGPase from L. major was subcloned into the His tag expression vector pET-22b (Novagen). Crystals of UGPase in complex with UDP-glucose were obtained from 27% PEG monomethyl ether 2000, 100 mM BisTris, pH 6.5. Phase Determination and Structure Refinement—The structure of the UGPase ϩ UDP-glucose complex was solved by the multiwavelength anomalous dispersion phasing method with programs from the CCP4 collection [14]. ARP/wARP including the warpNtrace protocol [16] were used to obtain a first glycine trace of the protein structure Starting from this initial model the complete catalytic domain and most of the N-terminal domain of the model could be built. In the apo structure two peaks of electron density are located at the active site and at the protein surface, which apparently are sulfate ions (SO42Ϫ) from the screening buffer. The final model of the ligand structure contained all residues beside the first and the last six residues of the full-length protein. The ligand UDPglucose was built into the model with the automated ligand
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
