High-resolution crystal structure of Trypanosoma brucei UDP-galactose 4′-epimerase: a potential target for structure-based development of novel trypanocides

Shaw, M.; Bond, Charles S.; Roper, Janine R.; Gourley, David G.; Ferguson, Michael A. J.; Hunter, William N.

doi:10.1016/s0166-6851(02)00243-8

Cited by 55 publications

(39 citation statements)

References 25 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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“…The presence of the hydrophobic Ala in C83A would not interfere with this hydrophobic interaction or produce electrostatic repulsion but should abolish hydrogen-bonding of the sulfydryl group of Cys83 with the 3 -hydroxyl group on the nicotinamide ribose of the bound NAD + , which is reflected by the dramatic increases in the cofactor and substrate K m values. Another member of the SCOR family, UDP-galacose 4-epimerase from Trypanosoma brucei, has been crystallized that has a similarly positioned Cys99 residue within the NAD + binding pocket, which may contribute to the stability of the substrate through van der Waals forces [22].…”

Section: Discussionmentioning

confidence: 99%

Structure/function of human type 1 3β-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

Thomas

Huether

Mack

et al. 2007

The Journal of Steroid Biochemistry and Molecular Biology

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The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.

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“…The latter can interconvert UDP-GlcNAc and UDP-GalNAc, whereas the trypanosome enzymes cannot (95,97). Second, high-resolution crystal structures of the T. brucei epimerase, and comparison with those of the human counterpart (44), suggest that there is potential for selective inhibition of the parasite enzyme (4,105). Third, a recent small-scale screen of 880 drug-like molecules revealed three compounds with 50% inhibitory concentration values for T. brucei GalE four times lower than those for human GalE (119).…”

Section: Cruzi) Gdp-man Is Also the Precursor Of Gdp-fuc (See Below)mentioning

confidence: 99%

Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

2007

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The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. The trypanosomatids are protozoan parasites that impose a major health burden on many countries in the developing world, causing a wide range of diseases over three continents. Like most eukaryotes, the cell surfaces and endosomal/lysosomal systems of these organisms are rich in glycoconjugates, some of which play essential roles in their survival, infectivity, or virulence. The glycoconjugate repertoires of the three trypanosomatids studied here, Trypanosoma brucei, T. cruzi, and Leishmania major, are fundamentally different, reflecting their disparate life cycles, modes of infection, and disease pathologies (see references 5,27,28,38,47,65, and 69 and references therein). The monosaccharides that make up these glycoconjugates vary between the three trypanosomatid species. For example, all three contain D-mannose (Man), D-Nacetylglucosamine (GlcNAc), D-glucosamine (GlcN), D-glucose (Glc), and D-galactopyranose (Galp), while only T. cruzi and L. major contain D-galactofuranose (Galf), only T. cruzi contains D-xylose (Xyl), L-rhamnopyranose (Rha), and L-fucose (Fuc), and only L. major contains D-arabinopyranose (Ara) ( Table 1). However, a unifying theme of their glycobiology is an abundance of cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and/or non-proteinlinked GPI structures.The protein-linked GPI glycans of the leishmania are the simplest in structure, consisting of the conserved Man␣1-2Man␣1-6Man␣1-4GlcN core. Those of T. cruzi are principally Man␣1-2Man␣1-2Man␣1-6Man␣1-4GlcN, and those of T. brucei are the most complex, with ␣-D-Galp and ␤-D-Galp side chains in the bloodstream form and sialylated poly-N-acetyllactosamine (poly-LacNAc) side chains in the procyclic form (27).The non-protein-linked GPI structures include the so-called glycoinositolphospholipids, or GIPLs, that are most abundant in the leishmania and T. cruzi species (58, 64, 90) but are also found in procyclic-form T. brucei (60,73,122) and bloodstream form T. brucei (39,63). Non-protein-linked GPIs also include the more exotic leishmania-specific lipophosphoglycan (LPG) structures (38,47,64,65). The leishmania LPGs contain characteristic phosphosaccharide repeats of Galp␤1-4Man␣1-P, which in L. major can have ␤-D-Galp and ␣-D-Arap side chains (66,67). These repeats are also found in the secr...

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High-resolution crystal structure of Trypanosoma brucei UDP-galactose 4′-epimerase: a potential target for structure-based development of novel trypanocides

Cited by 55 publications

References 25 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

Structure/function of human type 1 3β-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

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