Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB

Kronen, Miriam; Berg, Ivan A.

doi:10.1371/journal.pone.0145098

Cited by 17 publications

(34 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The growth phenotype of ∆fumACB on R, R-tartrate and itaconate could arise from PfFH playing a secondary but critical role required for cell growth. These results show that the substrate promiscuity profile of PfFH is similar to class I enzymes from other organisms (7,9,14,35) with the order of preference being fumarate followed by mesaconate and the least preferred being Dtartrate. However, the significance of the extended substrate specificity of PfFH for mesaconate and D-tartrate with regard to Plasmodium cellular biochemistry is unclear at this stage.…”

supporting

confidence: 54%

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

supporting

confidence: 54%

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

Section: Pffh Complements Fumarase Deficiency In E Coli-supporting

confidence: 55%

“…Meso-tartrate has two chiral carbons with S-configuration at C2, and R-configuration in C3. It should be noted that in the case of both EcFumC and EcFumA S, S-tartrate (D-tartrate) is a substrate (14). The inhibition by meso-tartrate of both class I and II FH indicates relaxed stereospecificity of these enzymes at the C3 carbon of the substrates.…”

Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB

Cited by 17 publications

References 67 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