Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacterium tuberculosis

Ruecker, Nadine; Jansen, Robert S.; Trujillo, Carolina; Puckett, Susan; Jayachandran, Pradeepa; Piroli, Gerardo G.; Frizzell, Norma; Molina, Henrik; Rhee, Kyu Y.; Ehrt, Sabine

doi:10.1016/j.chembiol.2017.01.005

Cited by 46 publications

(39 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Apart from perturbing AMP synthesis, high levels of fumarate can result in succination of cysteinyl residues in proteins and glutathione (51) thereby, compromising cellular homeostasis (52). Succinated proteome in human cell lines (53)(54)(55) and Mycobacterium tuberculosis (56) have been examined and these studies highlight the toxic effects of high levels of fumarate. Fumarate is recycled to aspartate through the action of enzymes FH, MQO and AAT.…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 99%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Apart from perturbing AMP synthesis, high levels of fumarate can result in succination of cysteinyl residues in proteins and glutathione (49) thereby, compromising cellular homeostasis (50). Succinated proteome in human cell lines (51)(52)(53) and Mycobacterium tuberculosis (54) have been examined and these studies highlight the toxic effects of high levels of fumarate. Fumarate is recycled to aspartate through the action of enzymes, FH, MQO and AAT.…”

Section: Resultsmentioning

confidence: 99%

Biochemical characterization and essentiality ofPlasmodiumfumarate hydratase

Jayaraman

Suryavanshi

Kalale

et al. 2017

Preprint

View full text Add to dashboard Cite

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. Fumarate, generated in large quantities in the parasite as a byproduct of AMP synthesis is converted to malate by the action of FH, and subsequently used in the generation of the key metabolites oxaloacetate, aspartate and pyruvate. 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. The substrate analog, mercaptosuccinic acid was found to be a potent inhibitor of PfFH with a K i value in the nanomolar range. Knockout of the fh gene was not possible in P. berghei when drug-selection of the transfectants was performed in BALB/c mice while the gene was amenable to knockout when C57BL/6 mice were used as host, thereby indicating mouse-strain dependent essentiality of the fh gene to the parasite.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intraerythrocytic 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 important candidate for further investigation.Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/158956 doi: bioRxiv preprint first posted onli...

show abstract

“…In most of these cases, succination lead to an irreversible inactivation of the protein activity. Recently, a fumarase deficient M. tuberculosis strain is shown to undergo fumarate‐mediated succination on cellular proteins, few of which were identified . The study demonstrates that fumarate accumulation (due to fumarase deficiency) leads to substantial loss of Mtb viability under infectious conditions.…”

Section: Discussionmentioning

confidence: 95%

“…The study demonstrates that fumarate accumulation (due to fumarase deficiency) leads to substantial loss of Mtb viability under infectious conditions. These studies show the regulation of cellular proteins via fumarate‐mediated succination and the phenomenon is demonstrated in M. tuberculosis as well . Since fumarate is one of the products in Mt ArgH catalysis, we hypothesized that the cysteine (Cys 441 ) present on Mt ArgH might undergo fumarate‐dependent modification as the reaction progresses.…”

Section: Discussionmentioning

confidence: 98%

Biochemical characterization of argininosuccinate lyase from M. tuberculosis: significance of a c‐terminal cysteine in catalysis and thermal stability

Mishra

2017

IUBMB Life

View full text Add to dashboard Cite

Arginine biosynthesis pathway is crucial to the survival and pathogenesis of Mycobacterium tuberculosis (Mtb). Arginine is a critical amino acid that contributes to the inflection of cellular immune responses during pathogenesis. Argininosuccinate lyase from Mtb (MtArgH), the last enzyme in the pathway, catalyzes the production of arginine from argininosuccinic acid. MtArgH is an essential enzyme for the growth and survival of M. tuberculosis. We biochemically characterized MtArgH and deciphered the role of a previously unexplored cysteine (Cys ) residue at the C-terminal region of the protein. Chemical modification of Cys completely abrogated the enzymatic activity suggesting its involvement in the catalytic mechanism. Replacement of Cys to alanine showed a striking decrease in the enzymatic activity, while retaining the overall secondary to quaternary structure of the protein, hence corroborating the involvement of Cys in the process of catalysis. Interestingly, replacement of Cys to serine, showed significant increase in activity, as compared to the wild-type MtArgH. Inactivity of C A and elevated activity of its conservative mutant (C S) confirmed the participation of Cys in the MtArgH activity. We also, observed that C S mutant has higher thermal stability and maintains significant activity at high temperatures. This is in concordance with our observation that Cys in Mtb is replaced by a serine in the ArgH from thermophilic microorganisms. Furthermore, we also propose a potential feedback mechanism, wherein the Cys is covalently modified to S-(2-succinyl) cysteine (succination) by one of the products, fumarate, thereby inactivating MtArgH. These insights into the mechanism of MtArgH activity unravel novel regulations of arginine biosynthetic pathway in Mtb. © 2017 IUBMB Life, 69(11):896-907, 2017.

show abstract

Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacterium tuberculosis

Cited by 46 publications

References 49 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality ofPlasmodiumfumarate hydratase

Biochemical characterization of argininosuccinate lyase from M. tuberculosis: significance of a c‐terminal cysteine in catalysis and thermal stability

Contact Info

Product

Resources

About