Galactose metabolism is essential for the African sleeping sickness parasite
            <i>Trypanosoma brucei</i>

Roper, Janine R.; Güther, Maria Lucia S.; Milne, Kenneth G.; Ferguson, Michael A. J.

doi:10.1073/pnas.092669999

Cited by 100 publications

(123 citation statements)

References 27 publications

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“…A UDP-hexose 4-epimerase from Trypanosoma brucei, a protozoan causing a variety of tropical diseases, has been reported to be essential for survival of its host. 23,24 Thus, a deeper understanding of substrate recognition by UDP-hexose 4-epimerases has potential implications in structure-based development of novel antibiotics. In addition, these results may also provide useful insights regarding other 4-epimerases.…”

Section: Discussionmentioning

confidence: 99%

Altered architecture of substrate binding region defines the unique specificity of UDP‐GalNAc 4‐epimerases

et al. 2011

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UDP-hexose 4-epimerases play a pivotal role in lipopolysaccharide (LPS) biosynthesis and Leloir pathway. These epimerases are classified into three groups based on whether they recognize nonacetylated UDP-hexoses (Group 1), both N-acetylated and nonacetylated UDPhexoses (Group 2) or only N-acetylated UDP-hexoses (Group 3). Although the catalysis has been investigated extensively, yet a definitive model rationalizing the substrate specificity of all the three groups on a common platform is largely lacking. In this work, we present the crystal structure of WbgU, a novel UDP-hexose 4-epimerase that belongs to the Group 3. WbgU is involved in biosynthetic pathway of the unusual glycan 2-deoxy-L-altruronic acid that is found in the LPS of the pathogen Pleisomonas shigelloides. A model that defines its substrate specificity is proposed on the basis of the active site architecture. Representatives from all the three groups are then compared to rationalize their substrate specificity. This investigation reveals that the Group 3 active site architecture is markedly different from the ''conserved scaffold'' of the Group 1 and the Group 2 epimerases and highlights the interactions potentially responsible for the origin of specificity of the Group 3 epimerases toward N-acetylated hexoses. This study provides a platform for further engineering of the UDP-hexose 4-epimerases, leads to a deeper understanding of the LPS biosynthesis and carbohydrate recognition by proteins. It may also have implications in development of novel antibiotics and more economic synthesis of UDP-GalNAc and downstream products such as carbohydrate based vaccines.

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Section: Discussionmentioning

confidence: 99%

Altered architecture of substrate binding region defines the unique specificity of UDP‐GalNAc 4‐epimerases

et al. 2011

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“…Gel Filtration and Analytical Ultracentrifugation of Recombinant T. brucei UDP-Glc 4Ј-Epimerase-Bacterial expression and purification of recombinant epimerase have been described previously (24). Recombinant protein (100 g at 1 mg/ml in phosphate-buffered saline (PBS)) was loaded onto a Superdex 200 HR30 HPLC column (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…Bloodstream form T. brucei cells (strain 427, variant 221) were grown in HMI-9 medium (26) supplemented with 10% fetal calf serum at 37°C and 5% CO 2 as described in Ref. 24.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we describe the nature and subcellular location of T. brucei UDP-Glc 4Ј-epimerase, an essential enzyme required for galactose metabolism in bloodstream form T. brucei (24), and show that it is also essential for the in vitro growth of procyclic form T. brucei. We also describe the changes in the cell surface molecular architecture of procyclic form T. brucei when they undergo partial and complete galactose starvation by genetic manipulation.…”

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confidence: 99%

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The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

Roper¹,

Güther

MacRae³

et al. 2005

Journal of Biological Chemistry

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Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDPGlc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins, were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.

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“…1). Because the trypanosomatid parasites Trypanosoma brucei and Trypanosoma cruzi are unable to take up galactose from the environment (9,10), the UDP-Glc 4-epimerase is indispensable for biosynthesis of UDP-Gal and derived glycoconjugates in these organisms and is essential for their survival (11)(12)(13)(14). In contrast, a salvage pathway for UDP-Gal synthesis is known to occur in Leishmania because radiolabeled Gal is taken up by promastigotes and incorporated into surface molecules (15).…”

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confidence: 99%

Leishmania UDP-sugar Pyrophosphorylase

Damerow

Lamerz

Haselhorst

et al. 2010

Journal of Biological Chemistry

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The Leishmania parasite glycocalyx is rich in galactosecontaining glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

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Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei

Cited by 100 publications

References 27 publications

Altered architecture of substrate binding region defines the unique specificity of UDP‐GalNAc 4‐epimerases

Altered architecture of substrate binding region defines the unique specificity of UDP‐GalNAc 4‐epimerases

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

Leishmania UDP-sugar Pyrophosphorylase

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