CSGID Solves Structures and Identifies Phenotypes for Five Enzymes in Toxoplasma gondii

Lykins, Joseph; Filippova, E.V.; Halavaty, A.S.; Minasov, G.; Zhou, Ying; Dubrovska, I.; Flores, K.; Shuvalova, L.; Ruan, Jiapeng; Bissati, Kamal El; Dovgin, Sarah; Roberts, Craig W.; Woods, Stuart; Moulton, Jon D.; Moulton, Hong M.; McPhillie, Martin J.; Muench, Stephen P.; Fishwick, Colin W. G.; Sabini, Elisabetta; Shanmugam, Dhanasekaran; Roos, David S.; McLeod, Rima; Anderson, W.F.; Ngô, Huân M.

doi:10.3389/fcimb.2018.00352

Cited by 14 publications

(14 citation statements)

References 108 publications

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“…The biophysical studies revealed that Ld RPE possesses adequate secondary structural elements with a predominately alpha helical content at physiological pH, which is in accordance with other leishmanial enzymes including PP1 27 and PepT. 28 Remarkably, the secondary structure observed in Ld RPE was comparable to crystal structures of RPEs from P. falciparum , 11 T. gondii , 29 and H. sapiens . 8 The melting temperature ( T m ) of Ld RPE has indicated cooperative unfolding, which is similar to that of previously reported in leishmanial protein phosphatase 2C.…”

Section: Discussionsupporting

confidence: 69%

Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani

Narsimulu

Qureshi²,

Jakkula

et al. 2021

ACS Omega

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Pentose phosphate pathway (PPP) plays a crucial role in the maintenance of NADPH/NADP + homeostasis and provides protection against oxidative stress through detoxification of the reactive oxygen species. Ribulose-5-phosphate epimerase (RPE) participates in catalysis of the interconversion of ribulose-5-phosphate (Ru5P) to xylulose-5-phosphate (Xu5P) during PPP, however the structural attributes of this enzyme are still underexplored in many human pathogens including leishmanial parasites. The present study focuses upon cloning, purification and characterization of RPE of Leishmania donovani ( Ld RPE) using various biophysical and structural approaches. Sequence analysis has shown the presence of trypanosomatid-specific insertions at the N-terminus that are absent in humans and other eukaryotes. Gel filtration chromatography indicated recombinant Ld RPE to exist as a dimer in the solution. Circular dichroism studies revealed a higher alpha helical content at physiological pH and temperature that comparatively varies with changing these parameters. Additionally, intrinsic fluorescence and quenching studies of Ld RPE have depicted that tryptophan residues are mainly buried in the hydrophobic regions, and the recombinant enzyme is moderately tolerant to urea. Moreover, homology modeling was employed to generate the three-dimensional structure of Ld RPE followed by molecular docking with the substrate, product, and substrate analogues. The modeled structure of Ld RPE unravelled the presence of conserved active site residues as well as a single binding pocket for the substrate and product, while an in silico study suggested binding of substrate analogues into a similar pocket with more affinity than the substrate. Additionally, molecular dynamics simulation analysis has deciphered complexes of Ld RPE with most of the ligands exhibiting more stability than its apo form and lesser fluctuations in active site residues in the presence of ligands. Altogether, our study presents structural insights into leishmanial RPE that could provide the basis for its implication to develop potent antileishmanials.

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Section: Discussionsupporting

confidence: 69%

Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani

Narsimulu

Qureshi²,

Jakkula

et al. 2021

ACS Omega

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“…Both reactions generate NADPH which is required as a reducing equivalent by other metabolic pathways including fatty acid synthesis [6,21]. The resulting Ru5P is then catalyzed to ribose 5-phosphate (R5P) and xylulose-5-phosphate (Xu5P) by ribose 5-phosphate isomerase (TgRPI) and ribulose 5-phosphate 3-epimerase (TgRuPE), respectively [17,22]. The non-oxidative branch of PPP in T. gondii comprises a set of reactions in which R5P and X5P are converted into fructose 6-phosphate (F6P) and glyceraldehyde 3-phosphate (GA3P) by transketolase (TgTKT) and transaldolase (TgTAL) [17].…”

Section: Introductionmentioning

confidence: 99%

Metabolic flexibilities and vulnerabilities in the pentose phosphate pathway of the zoonotic pathogen Toxoplasma gondii

Xia

Guo

et al. 2022

PLoS Pathog

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Metabolic pathways underpin the growth and virulence of intracellular parasites and are therefore promising antiparasitic targets. The pentose phosphate pathway (PPP) is vital in most organisms, providing a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose sugar for nucleotide synthesis; however, it has not yet been studied in Toxoplasma gondii, a widespread intracellular pathogen and a model protozoan organism. Herein, we show that T. gondii has a functional PPP distributed in the cytoplasm and nucleus of its acutely-infectious tachyzoite stage. We produced eight parasite mutants disrupting seven enzymes of the PPP in T. gondii. Our data show that of the seven PPP proteins, the two glucose-6-phosphate dehydrogenases (TgG6PDH1, TgG6PDH2), one of the two 6-phosphogluconate dehydrogenases (Tg6PGDH1), ribulose-5-phosphate epimerase (TgRuPE) and transaldolase (TgTAL) are dispensable in vitro as well as in vivo, disclosing substantial metabolic plasticity in T. gondii. Among these, TgG6PDH2 plays a vital role in defense against oxidative stress by the pathogen. Further, we show that Tg6PGDH2 and ribulose-5-phosphate isomerase (TgRPI) are critical for tachyzoite growth. The depletion of TgRPI impairs the flux of glucose in central carbon pathways, and causes decreased expression of ribosomal, microneme and rhoptry proteins. In summary, our results demonstrate the physiological need of the PPP in T. gondii while unraveling metabolic flexibility and antiparasitic targets.

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“…RPE can play a vital role in the development of a pool of NADPH, in the PPP that can convert monosaccharides, such as glucose, into the nucleotide precursor of pentose sugars. In addition, RPE can convert the ribulose-5-phosphate into xylulose 5-phosphate in the Calvin cycle [34]. RPE was identified with reversibly oxidized cysteines, which was another enzyme that uses ribulose-5-phosphate as the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, RPE was up-regulated significantly, which suggests that PPP was significantly active in the treatment group. This pathway has been suggested as a drug target in trypanosomes [34].…”

Section: Discussionmentioning

confidence: 99%

Label-Free Proteomic Analysis of Molecular Effects of 2-Methoxy-1,4-naphthoquinone on Penicillium italicum

Guo

Zhang

et al. 2019

IJMS

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Penicillium italicum is the principal pathogen causing blue mold of citrus. Searching for novel antifungal agents is an important aspect of the post-harvest citrus industry because of the lack of higher effective and low toxic antifungal agents. Herein, the effects of 2-methoxy-1,4-naphthoquinone (MNQ) on P. italicum and its mechanism were carried out by a series of methods. MNQ had a significant anti-P. italicum effect with an MIC value of 5.0 µg/mL. The label-free protein profiling under different MNQ conditions identified a total of 3037 proteins in the control group and the treatment group. Among them, there were 129 differentially expressed proteins (DEPs，up-regulated > 2.0-fold or down-regulated < 0.5-fold, p < 0.05), 19 up-regulated proteins, 26 down-regulated proteins, and 67 proteins that were specific for the treatment group and another 17 proteins that were specific for the control group. Of these, 83 proteins were sub-categorized into 23 hierarchically-structured GO classifications. Most of the identified DEPs were involved in molecular function (47%), meanwhile 27% DEPs were involved in the cellular component and 26% DEPs were involved in the biological process. Twenty-eight proteins identified for differential metabolic pathways by KEGG were sub-categorized into 60 classifications. Functional characterization by GO and KEGG enrichment results suggests that the DEPs are mainly related to energy generation (mitochondrial carrier protein, glycoside hydrolase, acyl-CoA dehydrogenase, and ribulose-phosphate 3-epimerase), NADPH supply (enolase, pyruvate carboxylase), oxidative stress (catalase, glutathione synthetase), and pentose phosphate pathway (ribulose-phosphate 3-epimerase and xylulose 5-phosphate). Three of the down-regulated proteins selected randomly the nitro-reductase family protein, mono-oxygenase, and cytochrome P450 were verified using parallel reaction monitoring. These findings illustrated that MNQ may inhibit P. italicum by disrupting the metabolic processes, especially in energy metabolism and stimulus response that are both critical for the growth of the fungus. In conclusion, based on the molecular mechanisms, MNQ can be developed as a potential anti-fungi agent against P. italicum.

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CSGID Solves Structures and Identifies Phenotypes for Five Enzymes in Toxoplasma gondii

Cited by 14 publications

References 108 publications

Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani

Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani

Metabolic flexibilities and vulnerabilities in the pentose phosphate pathway of the zoonotic pathogen Toxoplasma gondii

Label-Free Proteomic Analysis of Molecular Effects of 2-Methoxy-1,4-naphthoquinone on Penicillium italicum

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