Properties of GDP-mannose pyrophosphorylase, a critical enzyme and drug target in Leishmania mexicana

Davis, AJ; Perugini, MA; Smith, BJ; Stewart, JD; Ilg, T; Hodder, AN; Handman, E

Author(s): Davis, AJ; Perugini, MA; Smith, BJ; Stewart, JD; Ilg, T; Hodder, AN; Handman, E;
Details: Publication Year 2004-03-26,Volume 279,Issue #13,Page 12462-12468
Journal Title: JOURNAL OF BIOLOGICAL CHEMISTRY
Publication Type: Journal Article
Abstract: 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.
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Keywords: VIRULENCE; PHENOTYPES; PROTEINS
Publisher's Version: https://doi.org/10.1074/jbc.M312365200
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2004-03-26 12:00:00