Molecular Cloning of the Leishmania major UDP-glucose Pyrophosphorylase, Functional Characterization, and Ligand Binding Analyses Using NMR Spectroscopy

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Title Molecular Cloning of the Leishmania major UDP-glucose Pyrophosphorylase, Functional Characterization, and Ligand Binding Analyses Using NMR Spectroscopy
Author Lamerz, Anne-Christin; Haselhorst, Thomas Erwin; Bergfeld, Ann; von Itzstein, Mark; Gerardy-Schahn, Rita
Journal Name Journal of Biological Chemistry
Year Published 2006
Place of publication Bethesda, USA
Publisher The American Society for Biochemistry and Molecular Biology, Inc.
Abstract The dense glycocalyx surrounding the protozoan parasite Leishmania is an essential virulence factor. It protects the parasite from hostile environments in the sandfly vector and mammalian host and supports steps of development and invasion. Therefore, new therapeutic concepts concentrate on disturbing glycocalyx biosynthesis. Deletion of genes involved in the metabolism of galactose and mannose have been shown to drastically reduce Leishmania virulence. Here we report the identification of Leishmania major UDP-glucose pyrophosphorylase (UGP). UGP catalyzes the formation of UDP-glucose from glucose 1-phosphate and UTP. This activation step enables glucose to enter metabolic pathways and is crucial for the activation of galactose. UDP-galactose is made from UDP-glucose by nucleotide-donor transfer to galactose 1-phosphate or by epimerization of the glucose moiety. Isolated in a complementation cloning approach, the activity of L. major UGP was proven in vitro. Moreover, purified protein was used to investigate enzyme kinetics, quaternary organization, and binding of ligands. Whereas sequestration by oligomerization is a known regulatory mechanism for eukaryotic UGPs, the recombinant as well as native L. major UGP migrated as monomer in size exclusion chromatography and in accord with this showed simple Michaelis-Menten kinetics toward all substrates. In saturation transfer difference (STD)-NMR studies, we clearly demonstrated that the molecular geometry at position 4 of glucose is responsible for substrate specificity. Furthermore, the γ-phosphate group of UTP is essential for binding and for induction of the open conformation, which then allows entry of glucose 1-phosphate. Our data provide the first direct proof for the ordered bi-bi mechanism suggested in earlier studies.
Peer Reviewed Yes
Published Yes
Publisher URI http://www.jbc.org/
Alternative URI http://dx.doi.org/10.1074/jbc.M600076200
Volume 281
Issue Number 24
Page from 16314
Page to 16322
ISSN 0021-9258
Date Accessioned 2007-02-23
Date Available 2009-10-19T05:22:55Z
Language en_AU
Research Centre Institute for Glycomics
Faculty Institute for Glycomics
Subject PRE2009-Biochemistry and Cell Biology; PRE2009-Biophysics; PRE2009-Enzymes
URI http://hdl.handle.net/10072/13714
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1

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