Characterisation of the fumarate hydratase repertoire in Trypanosoma cruzi

Pádua, Ricardo Augusto Pereira de; Kia, Ali Martin; Costa-Filho, Antonio J.; Wilkinson, Shane R.; Nonato, Maria Cristina

doi:10.1016/j.ijbiomac.2017.03.099

Cited by 16 publications

(22 citation statements)

References 42 publications

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“…This provides a framework for developing class I PfFH (and in general for class I FH) specific inhibitors that will have no effect on the human enzyme. A recent study has reported the inhibition of TcFH by DL-MSA with a K i value of 4.2 ± 0.5 µM while no effect was observed on the class II human FH (22). The reason for DL-MSA's high specificity for class I FH must stem from the presence of 4Fe-4S cluster that interacts with the C2-hydroxyl group of malate (11).…”

Section: Discussionmentioning

confidence: 98%

“…The UV-visible spectrum with absorption maxima at 360 and 405 nm indicates the presence of 4Fe-4S cluster in the enzyme and 78 % reconstitution efficiency using an Ɛ value of 16,000 M -1 cm -1 at 410 nm (30). The addition of sodium dithionite lowered the absorption intensity at 405 nm indicating a reduction of the cluster (Fig 3b) (31)(32)(33)(34) (21) and Trypanosoma cruzi (22) while for those from bacteria and archaea, the values are in the micromolar range. The catalytic efficiency (k cat /K m ) of PfFH∆40 is similar to L. major FH but 10-100 fold lower than that reported for other class I FHs ( Table 2).…”

Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 95%

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Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a wellknown reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium. Fumarate is generated in large quantities in the parasite as a byproduct of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterizations of PfFH that may provide leads for the development of specific inhibitors are lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a K i value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain dependent.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intra-erythrocytic asexual stages, derives ATP primarily from glycolysis with low contribution from mitochondrial pathways (1, 2). The bulk of pyruvate formed is converted to lactic acid with a minor amount entering the tricarboxylic acid (TCA) cycle, the flux through which is upregulated in sexual stages (2). Key intermediates that anaplerotically feed into the TCA cycle are α-ketoglutarate derived from glutamate, oxaloacetate (OAA) from phosphoenolpyruvate, and fumarate from adenosine 5΄-monophosphate (AMP) synthesis. Synthesis of AMP in the parasite is solely from inosine-5΄-monophosphate (IMP) through a pathway involving the enzymes adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL). The net reaction of ADSS and ASL involves consumption of GTP and aspartate and, generation of GDP, P i and fumarate. In the rapidly dividing parasite with an AT-rich genome and high energy requirements, leading to a high demand for adenine pools, one would expect a high flux of fumarate generation. The parasite does not secrete fumarate but instead, the carbon derived from this metabolite can be traced in malate, OAA, aspartate, pyruvate (through PEP) and lactate (3). The metabolic significance of this fumarate anaplerosis is still obscure. In this context, fumarate hydratase (FH, fumarase) the key enzyme to metabolize fumarate becomes an Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon...

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Section: Discussionmentioning

confidence: 98%

Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 95%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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“…Abundant interactions for ribosomal proteins might be suggestive of the rapid and extensive protein translation that accompanies parasite differentiation and multiplication following host-cell invasion [15]. The ironsulfur cluster fumarase activity has been shown to be essential for protozoan parasites such as Trypanossoma cruzi since its mitochondrial isoform is part of the tricarboxylic acid cycle and as such being central to aerobic respiration [28]. The TCP1 complex belongs to the HSP60 family protein that was detected in P.…”

Section: Discussionmentioning

confidence: 99%

Shotgun Label-Free Proteomic Analyses of the Oyster Parasite Perkinsusmarinus

Leibowitz

Tavares

Pereira

et al. 2017

JPGR

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Perkinsus marinus is an intracellular parasitic protozoan that is responsible for serious disease epizootics in marine bivalve molluscs worldwide. Despite all available information on P. marinus genomics, more baseline data is required at the proteomic level. Our aim was to study the proteome profile of in vitro cultured P. marinus isolated from oysters Crassostrea spp. using a label-free shotgun UDMS E approach. A total of 4073 non redundant proteins were identified across three biological replicates with stringent identification. Proteins specifically related to adaptive survival, cell recognition, antioxidants, regulation of apoptosis and others were detected. Important virulence factors of P. marinus were identified including serine protease and irondependent superoxide dismutase. Other proteins with involvement in several pathogens invasion strategies were rhoptries, serine-threonine kinases and protein phosphatases. Interestingly, peptides corresponding to retroviruses polyproteins were identified in all replicates. The interactomic analysis of P. marinus proteins demonstrated important cluster networks related to biological processes. In conclusion, we provide the first comprehensive proteomic profile of P. marinus that can be useful for further investigations on Perkinsus biology and virulence mechanisms.

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“…Fumarases usually fall into two distinct classes according to their oligomeric state, sensitivity to oxygen and metal‐dependence . Class I fumarases are homodimeric (with a molecular weight of 120 kDa), contain an iron‐sulfur cluster (4Fe–4S) in the catalytic center and are oxygen‐sensitive . The class II fumarases are homotetrameric (with a molecular weight of about 200 kDa), are iron independent and carry no other cofactors .…”

Section: Introductionmentioning

confidence: 99%

Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase

et al. 2019

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Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l‐malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X‐ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221, with unit‐cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C‐terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES‐binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.

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Characterisation of the fumarate hydratase repertoire in Trypanosoma cruzi

Cited by 16 publications

References 42 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Shotgun Label-Free Proteomic Analyses of the Oyster Parasite Perkinsusmarinus

Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase

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