Disulfide Bridges in the Mesophilic Triosephosphate Isomerase from Giardia lamblia Are Related to Oligomerization and Activity

Reyes‐Vivas, Horacio; Dı́az, Adelaida; Peón, Jörge; Mendoza‐Hernández, Guillermo; Hernández-Alcántara, Gloria; Mora, Ignacio De la Mora-De la; Enríquez-Flores, Sergio; Domínguez-Ramírez, Lenin; López-Velázquez, Gabriel

doi:10.1016/j.jmb.2006.10.053

Cited by 29 publications

(43 citation statements)

References 74 publications

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“…Added to this, the VSPs of Giardia confer antigenic variation (1), and their overexpression is related to high virulence (44). Such conditions have driven our group to search for new antigiardial drugs, with a special interest in triosephosphate isomerase as a molecular target (22,24,31). Based on our previous data, we proposed omeprazole and its isomers (G. López-Ve- lázquez and H. Reyes-Vivas, 6 October 2008, Mexican Patent Office) as drugs with a potential to inhibit GlTIM and kill Giardia trophozoites.…”

Section: Discussionmentioning

confidence: 99%

“…Although omeprazole modifies 1 of its 5 Cys in the same way as when we used DTNB (22), it does not inactivate HsTIM; indeed, it is almost 3,000 times more resistant than GlTIM. This is due to the properties of the region surrounding Cys 222 in GlTIM and its influence on the affinity for the substrate by the enzyme when such a residue is modified (24,27,31). The fluorescence of GlTIM-omeprazole adducts observed in recombinant enzymes (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Giardia strains. G. lamblia WB strain was acquired from the American Type Culture Collection (ATCC), cultured, harvested, and maintained as previously described (31). Metronidazole resistance was induced in WB by conventional methods.…”

Section: Methodsmentioning

confidence: 99%

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Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

Reyes‐Vivas

Mora

Castillo-Villanueva

et al. 2014

Antimicrob Agents Chemother

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Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

Reyes‐Vivas

Mora

Castillo-Villanueva

et al. 2014

Antimicrob Agents Chemother

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“…3A ). The K m of LvTIM was 0.7 mM, higher than the K m reported for human TIM (0.49 mM) [5] and TIMs from protozoan parasites such as L. donovani (0.328 mM) [18], L. mexicana (0.30 mM) [37], G. lamblia (0.53 mM) [38], T. brucei (0.25 mM) [39], but lower than the K m from TIMs from Vibrio marinus (1.9 mM) [9] or S. cerevisiae (1.27 mM) [40]. The LvTIM k cat was 1.2 × 10 5 min −1 , similar to the k cat reported for other TIMs such L. mexicana (2.5 ×10 5 min −1 ) [37], Helicobacter pylori (8.8 ×10 4 min −1 ) [41], T. brucei (3.7 ×10 5 min −1 ) [39] and rabbit muscle TIM (5.1 ×10 5 min −1 ) [42].…”

Section: Resultsmentioning

confidence: 94%

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

López-Zavala

Carrasco-Miranda

Ramirez-Aguirre³

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation.

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“…As previously reported, the crystallization assays of GlTIM are conducted with the C202A mutant, which forms stable dimers that allows obtaining crystals suitable for diffraction [19]. The C202A mutation does not have appreciable effects on the catalytic properties or the susceptibility to cysteine derivatizants of GlTIM [19].…”

Section: Methodsmentioning

confidence: 99%

Structural and Functional Perturbation of Giardia lamblia Triosephosphate Isomerase by Modification of a Non-Catalytic, Non-Conserved Region

et al. 2013

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BackgroundWe have previously proposed triosephosphate isomerase of Giardia lamblia (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation.ResultsIn this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design.ConclusionsThe results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.

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Disulfide Bridges in the Mesophilic Triosephosphate Isomerase from Giardia lamblia Are Related to Oligomerization and Activity

Cited by 29 publications

References 74 publications

Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

Structural and Functional Perturbation of Giardia lamblia Triosephosphate Isomerase by Modification of a Non-Catalytic, Non-Conserved Region

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