Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase

Pádua, Ricardo Augusto Pereira de; Nonato, Maria Cristina

doi:10.1107/s2053230x13033955

Cited by 12 publications

(13 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Many organisms have genes corresponding to both the classes, as in Escherichia coli (Ec), which has three FH encoding genes viz., fum A, B and C. Fum A and fum B are 4Fe-4S cluster containing class I enzymes, while Fum C belongs to class II type FH. Recently, another gene fum D has been identified in the E. coli genome to code for a class I fumarase with altered substrate preferences (14).The structural and biochemical characteristics of class II FH are thoroughly studied from different organisms viz., human, porcine, yeast, E. coli and other sources (15)(16)(17)(18)(19). On the other hand, class I FH is not well studied owing to its thermolabile and oxygen sensitive nature.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

mentioning

confidence: 99%

“…The structural and biochemical characteristics of class II FH are thoroughly studied from different organisms viz., human, porcine, yeast, E. coli and other sources (15)(16)(17)(18)(19). On the other hand, class I FH is not well studied owing to its thermolabile and oxygen sensitive nature.…”

mentioning

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

“…Thus, a prospective tumor/normal sequencing study, which represent the current gold-standard (45), in a representative risk group is needed. Based on literature recommendations (44,57) and our experience with heritable tumor syndromes, a possible strategy would be to offer this tumor/normal screening to every symptomatic woman below age of 40 years who has multiple ULs or one UL (38). Green, Lyase domain; pale-green, FumC-C domain; red, amino acid residues for the mutations L315P, H196P, G275A, M412I, A273T, H196L lying in the Lyase domain or close to it.…”

Section: Different Screening Approaches Based On Morphological or Immmentioning

confidence: 99%

Targeted sequencing of FH-deficient uterine leiomyomas reveals biallelic inactivating somatic fumarase variants and allows characterization of missense variants

Popp

Erber

Kraus

et al. 2019

Preprint

View full text Add to dashboard Cite

Uterine leiomyomas (ULs) constitute a considerable health burden in the general female population. The fumarate hydratase (FH) deficient subtype is found in up to 1.6% of cases.Identifying these individuals is important as a subset of cases is related to germline FH variants in the setting of the hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome which is associated with aggressive renal cell cancer.We sequenced 13 FH-deficient ULs using the TruSight Cancer Panel. In all 13 cases, we could identify biallelic FH variants. In eight cases, we found a FH point mutation (two truncating, six missense) with evidence for loss of the second allele. Variant allelefrequencies pointed to somatic variants in all cases with a point mutation. Spatial clustering of the identified missense variants in the lyase domain indicated their importance for proper fumarase oligomerization. In five tumors, we found a biallelic deletion of FH. Analysis of driver mutations of other genes revealed two truncating TP53 variants in one case. Copy number (CN) analysis found a large RB1 deletion in three tumors and enrichment for ALK CN gains. Following our recommendation, one individual presented for genetic consultation. A germlineFH variant could not be identified. Clinical examination and family history raised the suspicion of tuberous sclerosis complex, which could subsequently be confirmed due to the splice variant c.2040A>G in TSC1. This case highlights that presence of FH deficiency does not exclude other concurrent genetic syndromes.By comparing the FH mutation carrier frequency in public databases and the prevalence of FH deficient ULs, we conclude that most of these are likely sporadic and estimate 3.5 -14.3% of females with an FH deficient UL to carry a germline FH variant. Further prospective tumor/normal sequencing studies are needed to develop a reliable screening strategy for HLRCC in women with ULs.

show abstract

“…Trypanosoma cruzi, both Kinetoplastids (18f, 19), and the 3-dimensional structure of LmFH II (11) are the only reports of class I FH from eukaryotes. All Plasmodium species have one gene annotated putatively as fumarate hydratase that remains to be characterized.…”

Section: Introductionmentioning

confidence: 99%

mentioning

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

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase

Cited by 12 publications

References 17 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Targeted sequencing of FH-deficient uterine leiomyomas reveals biallelic inactivating somatic fumarase variants and allows characterization of missense variants

Biochemical characterization and essentiality ofPlasmodiumfumarate hydratase

Contact Info

Product

Resources

About