Tight‐binding inhibitors efficiently inactivate both reaction centers of monomeric <i>Plasmodium falciparum</i> glyoxalase 1

Urscher, Miriam; More, Swati S.; Alisch, Romy; Vince, Robert; Deponte, Marcel

doi:10.1111/j.1742-4658.2012.08640.x

Cited by 21 publications

(21 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PCR primers were purchased from Metabion. N terminally MRGS(H) 6 -tagged wild type and mutant ScGrx7, PfGrx and PfGR were expressed in E. coli strain XL1-Blue and purified by affinity chromatography using an elution buffer containing 200 mM imidazole, 300 mM NaCl and 50 mM sodium phosphate, pH 8.0 (refs 11, 12, 18, 33, 53). Recombinant His-tagged AtGrxS15, AtGrxC1 and roGFP2 were also expressed and purified according to established protocols3436.…”

Section: Methodsmentioning

confidence: 99%

Glutaredoxin catalysis requires two distinct glutathione interaction sites

et al. 2017

Self Cite

View full text Add to dashboard Cite

Glutaredoxins are key players in cellular redox homoeostasis and exert a variety of essential functions ranging from glutathione-dependent catalysis to iron metabolism. The exact structure–function relationships and mechanistic differences among glutaredoxins that are active or inactive in standard enzyme assays have so far remained elusive despite numerous kinetic and structural studies. Here, we elucidate the enzymatic mechanism showing that glutaredoxins require two distinct glutathione interaction sites for efficient redox catalysis. The first site interacts with the glutathione moiety of glutathionylated disulfide substrates. The second site activates glutathione as the reducing agent. We propose that the requirement of two distinct glutathione interaction sites for the efficient reduction of glutathionylated disulfide substrates explains the deviating structure–function relationships, activities and substrate preferences of different glutaredoxin subfamilies as well as thioredoxins. Our model also provides crucial insights for the design or optimization of artificial glutaredoxins, transition-state inhibitors and glutaredoxin-coupled redox sensors.

show abstract

Section: Methodsmentioning

confidence: 99%

Glutaredoxin catalysis requires two distinct glutathione interaction sites

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…ATM was from Sciphar. Recombinant Pf AOP with an N-terminal MRGSH 6 GS-tag replacing the first 59 amino acid residues of the full length protein as well as P. falciparum glutathione reductase ( Pf GR) and P. falciparum glutaredoxin ( Pf Grx) were produced in Escherichia coli and purified by affinity chromatography as described previously 45, 72 . Plasmid pUF1-Cas9 for the expression of Cas9 and plasmid pL6 for guide RNA expression and homologous recombination in P. falciparum 53 were kind gifts from J.J. Lopez-Rubio.…”

Section: Methodsmentioning

confidence: 99%

Knockout of the peroxiredoxin 5 homologue PFAOP does not affect the artemisinin susceptibility of Plasmodium falciparum

Djuika

Staudacher

Sánchez

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

Artemisinins are the current mainstay of malaria chemotherapy. Their exact mode of action is an ongoing matter of debate, and several factors have recently been reported to affect an early stage of artemisinin resistance of the most important human malaria parasite Plasmodium falciparum. Here, we identified a locus on chromosome 7 that affects the artemisinin susceptibility of P. falciparum in a quantitative trait locus analysis of a genetic cross between strains 7G8 and GB4. This locus includes the peroxiredoxin gene PFAOP. However, steady-state kinetic data with recombinant PfAOP do not support a direct interaction between this peroxidase and the endoperoxide artemisinin. Furthermore, neither the overexpression nor the deletion of the encoding gene affected the IC50 values for artemisinin or the oxidants diamide and tert-butyl hydroperoxide. Thus, PfAOP is dispensable for blood stage parasite survival, and the correlation between the artemisinin susceptibility and chromosome 7 is probably based on another gene within the identified locus.

show abstract

“…Therefore, the increased glycolytic fluxes in T. gondii tachyzoites would suggest that these parasites require an efficient detoxification system for removal of harmful methylglyoxal. While there are several reports of studies focusing on the glyoxalase system of Plasmodium species as a potential drug target, no such study has been reported in T. gondii to date [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures

et al. 2015

View full text Add to dashboard Cite

BackgroundThe glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy.MethodsThe T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii.ResultsActive recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The Ki and IC50 were 12.9 ± 0.5 μM and 38.3 ± 0.9 μM, respectively.ConclusionThe inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin’s direct inhibition of the glyoxalase system of T. gondii will be needed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1268-5) contains supplementary material, which is available to authorized users.

show abstract

Tight‐binding inhibitors efficiently inactivate both reaction centers of monomeric Plasmodium falciparum glyoxalase 1

Cited by 21 publications

References 34 publications

Glutaredoxin catalysis requires two distinct glutathione interaction sites

Glutaredoxin catalysis requires two distinct glutathione interaction sites

Knockout of the peroxiredoxin 5 homologue PFAOP does not affect the artemisinin susceptibility of Plasmodium falciparum

Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures

Contact Info

Product

Resources

About