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J. Biol. Chem., Vol. 281, Issue 24, 16314-16322, June 16, 2006

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

Anne-Christin Lamerz, Thomas Haselhorst, Anne K. Bergfeld, Mark von Itzstein¹, and Rita Gerardy-Schahn²

From the Zelluläre Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany and the Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, 9726, Australia

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 -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.

Received for publication, January 4, 2006 , and in revised form, March 20, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ328944 [GenBank] .

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by an Australian Federation Fellowship of the Australian Research Council and by the National Health and Medical Research Council.

² Supported by Deutsche Forschungsgemeinschaft Grant Ge 801/4-1,2 and the Fonds der Chemischen Industrie. To whom correspondence should be addressed. Tel.: 49-511-532-9802; Fax: 49-511-532-8801; E-mail: gerardy-schahn.rita{at}mh-hannover.de.

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