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 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 alpha/beta 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.
Davis
Antony J
AJ
The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia.
Perugini
Matthew A
MA
Smith
Brian J
BJ
Stewart
James D
JD
Ilg
Thomas
T
Hodder
Anthony N
AN
Handman
Emanuela
E
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D007894:Leishmania mexicana / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009713:Nucleotidyltransferases / Q000737:chemistry*; D017830:Octoxynol; D010641:Phenotype; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D014867:Water / Q000737:chemistry
D000981:Antiprotozoal Agents; D002352:Carrier Proteins; D003902:Detergents; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D014867:Water; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; C023226:UDPacetylglucosamine pyrophosphorylase