Biology subjects

Suryavanshi, A.

Publications and source records attributed to Suryavanshi, A..

3 recordsLinked to original sources

bioRxiv2026

Perturbations in fumarate levels in Plasmodium berghei leads to cysteine succination and impairs ookinete formation

The TCA cycle intermediates comprise 8 carboxylic acids of which only fumarate has unsaturated carbons rendering it capable of electrophilic addition to cysteine thiols. Accumulation of fumarate due to loss of function of fumarate hydratase converts it into a powerful driver of cancer and therefore is classified as an oncometabolite. We have examined the consequences of perturbing the metabolism of fumarate and its product, malate, on Plasmodium berghei development across erythrocytic and early insect stages. Our studies on P. berghei lines lacking the genes fh and mqo, coding for the enzymes fumarate hydratase (FH) and malate quinone oxidoreductase (MQO), respectively as well as dtc and ogc coding for the transporters dicarboxylate-tricarboxylate carrier (DTC) and citrate-oxoglutarate carrier (OGC), show dramatic impairment in ookinete formation, while gametocytes and erythrocytic asexual stages remain largely unaffected. Comparative metabolomic analysis of gametocytes revealed that elevated fumarate levels in the knockouts led to succination of glutathione, possibly resulting in oxidative stress. The increased levels of the M+5 isotopologue of inosine monophosphate in the knockout gametocytes, observed in isotope tracer experiments, suggest enhanced ribose-5-phosphate and NADPH production through the pentose phosphate pathway, with the latter potentially mitigating elevated oxidative stress. The isotope tracer studies also informed that the P. berghei DTC is a transporter of malate and fumarate and OGC, a transporter of fumarate. The impaired ookinete formation arising from cysteine succination underscores the potential for developing transmission-blocking agents through selective inhibition of class I parasite FH which is distinct from its class II human counterpart. Significance statementMetabolic requirements of Plasmodium that lives across two hosts, traversing developmental stages with different rates of growth, are highly varied. Previous studies have established the non-essentiality of the TCA cycle genes for the erythrocytic asexual stages. Our studies show that knockout of genes involved in fumarate and malate metabolism, and transporters involved in anaplerosis, impairs development of the mosquito-stage, ookinete, with the gametocytes of the knockout lines exhibiting elevated levels of fumarate. The accumulation of succinated glutathione, formed via Michael addition of fumarate to the cysteinyl thiol group and leading to oxidative stress, is probably the leading cause of impairment in ookinete development.

biochemistry↗

bioRxiv2022

Revisiting the burden borne by fumarase: enzymatic hydration of an olefin

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol-1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, that have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large inter-subunit rearrangements occur across apo- and the holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis and computational studies including DFT and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH, polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/506457v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@f2f7feorg.highwire.dtl.DTLVardef@1774e6aorg.highwire.dtl.DTLVardef@1bd180eorg.highwire.dtl.DTLVardef@1764941_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2017

Biochemical characterization and essentiality of Plasmodium fumarate hydratase

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 well-known 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 Ki 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.

biochemistry↗