Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from
            <i>Leishmania major</i>
            reveals a unique protein fold

Feliciano, P.R.; Drennan, Catherine L.; Nonato, Maria Cristina

doi:10.1073/pnas.1605031113

Cited by 27 publications

(75 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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). Replacement of the hydroxyl group with a thiol probably leads to tight binding through Fe-S interaction.…”

Section: Discussionmentioning

confidence: 98%

“…Class I fumarases display substrate promiscuity; apart from catalyzing the interconversion of fumarate and malate, these enzymes also interconvert S, S-tartrate and oxaloacetate and, mesaconate and S-citramalate with varying catalytic efficiencies (9,10). The 4Fe-4S cluster is bound to the enzyme by 3 metalthiolate bonds formed between 3 conserved cysteine residues in the protein and 3 ferrous ions (11). The fourth iron in the cluster, proposed to be held loosely by a hydroxyl ion is thought to be directly involved in substrate binding and catalysis as seen in the enzyme aconitase (12,13).…”

mentioning

confidence: 99%

“…All Apicomplexans and Kinetoplastids possess only class I FH, whereas Dinoflagellates have both the classes (20). Biochemical characterization of class I FH from Leishmania major (Lm) and Trypanosoma cruzi, both Kinetoplastids (21,22), 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.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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: 98%

mentioning

confidence: 99%

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

show abstract

“…A recent study has reported the inhibition of TcFH by DL-MSA with a K i of 4.2 ± 0.5 µM while no effect was observed on the class II human FH (22). Interestingly, DL-MSA is not a substrate for PfFH as seen by spectrophotometric assays at 240 nm with 10 mM DL-MSA and 1 µM enzyme that failed to show either formation of the enediolate intermediate or the product fumarate through the liberation of H 2 S. 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). Replacement of the hydroxyl group with a thiol probably leads to tight binding through Fe-S interaction.…”

Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 98%

“…Class I fumarases display substrate promiscuity; apart from catalyzing the interconversion of fumarate and malate, these enzymes also interconvert S,S-tartrate and oxaloacetate and, mesaconate and S-citramalate with varying catalytic efficiencies (9,10). The 4Fe-4S cluster is bound to the enzyme by 3 metal-thiolate bonds formed between 3 conserved cysteine residues in the protein and 3 ferrous ions (11). The fourth iron in the cluster proposed to be held loosely by a hydroxyl ion, is thought to be directly involved in substrate binding and catalysis as seen in the enzyme aconitase (12,13).…”

Section: Introductionmentioning

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

Enantioselectivity in the enzymatic dehydration of malate and tartrate: Mirror image specificities of structurally similar dehydratases

Bellur,

Mukherjee,

Sharma

et al. 2023

Protein Science

View full text Add to dashboard Cite

Malate (2‐hydroxysuccinic acid) and tartrate (2,3‐dihydroxysuccinic acid) are chiral substrates; the former existing in two enantiomeric forms (R and S) while the latter exists as three stereoisomers (R,R; S,S; and R,S). Dehydration by stereospecific hydrogen abstraction and antielimination of the hydroxyl group yield the achiral products fumarate and oxaloacetate, respectively. Class‐I fumarate hydratase (FH) and L‐tartrate dehydratase (L‐TTD) are two highly conserved enzymes belonging to the iron–sulfur cluster hydrolyase family of enzymes that catalyze reactions on specific stereoisomers of malate and tartrate. FH from Methanocaldococcus jannaschii accepts only (S)‐malate and (S,S)‐tartrate as substrates while the structurally similar L‐TTD from Escherichia coli accepts only (R)‐malate and (R,R)‐tartrate as substrates. Phylogenetic analysis reveals a common evolutionary origin of L‐TTDs and two‐subunit archaeal FHs suggesting a divergence during evolution that may have led to the switch in substrate stereospecificity preference. Due to the high conservation of their sequences, a molecular basis for switch in stereospecificity is not evident from analysis of crystal structures of FH and predicted structure of L‐TTD. The switch in enantiomer preference may be rationalized by invoking conformational plasticity of the amino acids interacting with the substrate, together with substrate reorientation and conformer selection about the C2C3 bond of the dicarboxylic acid substrates. Although classical models of enzyme–substrate binding are insufficient to explain such a phenomenon, the enantiomer superposition model suggests that a minor reorientation in the active site residues could lead to the switch in substrate stereospecificity.

show abstract

Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold

Cited by 27 publications

References 35 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality ofPlasmodiumfumarate hydratase

Enantioselectivity in the enzymatic dehydration of malate and tartrate: Mirror image specificities of structurally similar dehydratases

Contact Info

Product

Resources

About