Gloria Hernández-Alcántara

Determining the molecular mechanism of inactivation by chemical modification of triosephosphate isomerase from the human parasite Giardia lamblia: a study for antiparasitic drug design.

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol‐reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol‐reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs. Proteins 2011;.

The Stability of G6PD Is Affected by Mutations with Different Clinical Phenotypes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The kcat (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T50 (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP+ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.

An unusual triosephosphate isomerase from the early divergent eukaryote Giardia lamblia

Recombinant triosephosphate isomerase from the parasite Giardia lamblia (GlTIM) was characterized and immunolocalized. The enzyme is distributed uniformly throughout the cytoplasm. Size exclusion chromatography of the purified enzyme showed two peaks with molecular weights of 108 and 55 kDa. Under reducing conditions, only the 55‐kDa protein was detected. In denaturing gel electrophoresis without dithiothreitol, the enzyme showed two bands with molecular weights of 28 and 50 kDa; with dithiotretitol, only the 28‐kDa protein was observed. These data indicate that GlTIM may exist as a tetramer or a dimer and that, in the former, the two dimers are covalently linked by disulfide bonds. The kinetics of the dimer were similar to those of other TIMs. The tetramer exhibited half of the kcat of the dimer without changes in the Km. Studies on the thermal stability and the apparent association constants between monomers showed that the tetramer was slightly more stable than the dimer. This finding suggests the oligomerization is not related to enzyme thermostability as in Thermotoga maritima. Instead, it could be that oligomerization is related to the regulation of catalytic activity in different states of the life cycle of this mesophilic parasite. Proteins 2004.

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

Background We 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. Results In 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. Conclusions The 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.

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

ABSTRACT 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.

Key Residues of Loop 3 in the Interaction with the Interface Residue at Position 14 in Triosephosphate Isomerase from Trypanosoma brucei

Cysteine 14 is an interface residue that is fundamental for the catalysis and stability of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM). Its side chain is surrounded by a deep pocket of 11 residues that are part of loop 3 of the adjacent monomer. Mutation of this residue to serine (producing single mutant C14S) yields a wild-type-like enzyme that is resistant to the action of sulfhydryl reagents methylmethane thiosulfonate (MMTS) and 5,5-dithiobis(2-nitrobenzoate) (DTNB). This mutant enzyme was a starting point for probing by cysteine scanning the role of four residues of loop 3 in the catalysis and stability of the enzyme. Considering that the conservative substitution of either serine or alanine with cysteine would minimally alter the structure and properties of the environment of the residue in position 14, we made double mutants C14S/A69C, C14S/S71C, C14S/A73C, and C14S/S79C. Three of these double mutants were similar in their kinetic parameters to wild-type TbTIM and the single mutant C14S, but double mutant C14S/A73C showed a greatly reduced k cat. All enzymes had similar CD spectra, but all mutants had thermal stabilities lower than that of wild-type TbTIM. Intrinsic fluorescence was also similar for all enzymes, but the double mutants bound up to 50 times more 1-anilino-8-naphthalene sulfonate (ANS) and were susceptible to digestion with subtilisin. The double mutants were also susceptible to inactivation by sulfhydryl reagents. Double mutant C14S/S79C exhibited the highest sensitivity to MMTS and DTNB, bound a significant amount of ANS, and had the highest sensitivity to subtilisin. Thus, the residues at positions 73 and 79 are critical for the catalysis and stability of TbTIM, respectively.

Unraveling the mechanisms of tryptophan fluorescence quenching in the triosephosphate isomerase from Giardia lamblia

In the native state several proteins exhibit a quenching of fluorescence of their tryptophans. We studied triosephosphate isomerase from Giardia lamblia (GlTIM) to dissect the mechanisms that account for the quenching of fluorescence of its Trp. GlTIM contains four Trp per monomer (Trp75, Trp162, Trp173, and Trp196) distributed throughout the 3D structure. The fluorescence of the denatured enzyme is 3-fold higher than that of native GlTIM. To ascertain the origin of this phenomenon, single and triple mutants of Trp per Phe were made. The intrinsic fluorescence was determined, and the data were interpreted on the basis of the crystal structure of the enzyme. Our data show that the fluorescence of all Trp residues is quenched through two different mechanisms. In one, fluorescence is quenched by aromatic-aromatic interactions due to the proximity and orientation of the indole groups of Trp196 and Trp162. The magnitude of the quenching of fluorescence in Trp162 is higher than in the other three Trp. Fluorescence quenching is also due to energy transfer to the charged residues that surround Trp 75, 173 and 196. Further analysis of the fluorescence of GlTIM showed that, among TIMs from other parasites, Trp at position 12 exhibits rather unique properties.

Crosstalk between the subunits of the homodimeric enzyme triosephosphate isomerase

Homodimeric triosephosphate isomerase (TIM) from Trypanosoma cruzi (TcTIM) and T. brucei (TbTIM) are markedly similar in amino acid sequence and three‐dimensional structure. In their dimer interfaces, each monomer has a Cys15 that is surrounded by loop3 of the adjoining subunit. Perturbation of Cys15 by methylmethane thiosulfonate (MMTS) induces abolition of catalysis and structural changes. In the two TIMs, the structural arrangements of their Cys15 are almost identical. Nevertheless, the susceptibility of TcTIM to MMTS is nearly 100‐fold higher than in TbTIM. To ascertain the extent to which the characteristics of the interface Cys depend on the dynamics of its own monomer or on those of the adjacent monomer, we studied MMTS action on mutants of TcTIM that had the interface residues of TbTIM, and hybrids that have only one interfacial Cys15 (C15ATcTIM‐wild type TbTIM). We found that the solvent exposure of the interfacial Cys depends predominantly on the characteristics of the adjoining monomer. The maximal inhibition of activity induced by perturbation of the sole interface Cys in the C15ATcTIM‐TbTIM hybrid is around 60%. Hybrids formed with C15ATcTIM monomers and catalytically inert TbTIM monomers (E168DTbTIM) were also studied. Their activity drops by nearly 50% when the only interfacial Cys is perturbed. These results in conjunction with those on C15ATcTIM‐wild type TbTIM hybrid indicate that about half of the activity of each monomer depends on the integrity of each of the two Cys15‐loop3 portions of the interface. This could be another reason of why TIM is an obligatory dimer. Proteins 2007;

New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent catalysis.

First characterization of a microsporidial triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable target

The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.