Properties of GDP-mannose Pyrophosphorylase, a Critical Enzyme and Drug Target in Leishmania mexicana

Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis...

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Veröffentlicht in:The Journal of biological chemistry 2004-03, Vol.279 (13), p.12462-12468
Hauptverfasser: Davis, Antony J., Perugini, Matthew A., Smith, Brian J., Stewart, James D., Ilg, Thomas, Hodder, Anthony N., Handman, Emanuela
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Sprache:eng
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Zusammenfassung:Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed α/β structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M312365200