Biology subjects

Balaram, H.

Publications and source records attributed to Balaram, H..

13 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↗

bioRxiv2025

Insights into the mechanism of succinimide formation in an archaeal glutaminase.

Succinimide (SNN), an intermediate formed during asparaginyl deamidation or aspartyl dehydration in proteins, is generally hydrolysis-prone, leading to isomerization to L/D /{beta}-aspartyl residue, with the latter being considered deleterious to protein structure and function. An unusually stable SNN-mediated conformational rigidity through restriction of the backbone dihedral angle, {psi}, enhances the thermostability of glutamine amidotransferase (GATase) from Methanocaldococcus jannaschii (Mj). Although several structural features involved in maintaining a stable SNN and imparting SNN-mediated thermostability have been identified in MjGATase, the residues in the protein that catalyse the rapid and complete conversion of Asn109 to SNN remain unknown. Here, we investigated several site-directed mutants of MjGATase for their ability to retain Asn109 side chain in the unmodified form. Mass spectrometric analysis of 10 single mutants enabled the identification of residues that impacted the proportion of SNN and Asn population in the protein sample. This led to the generation of two double mutants that retained intact Asn109 side chain as observed in the mass spectra and crystal structures. These mutants with intact Asn residue at position 109, displayed lower thermal stability than the protein with the SNN modification. Further understanding of the deprotonation mechanism was addressed using QM/MM MD metadynamics simulations. HighlightsO_LIStable succinimide (SNN) arising from deamidation of Asn109 residue imparts hyperthermostability to MjGATase. C_LIO_LIExamination of the structure of MjGATase suggests neighbouring residues playing possible roles in deamidation and cyclization. C_LIO_LIExamination by LC-MS of single site-directed mutants of residues contacting SNN revealed varied levels of intact Asn109 enabling generation of double mutants with complete absence of deamidation. C_LIO_LIPresence of intact Asn109 confirmed by X-ray crystallography highlights the role of Y158, D110, and K151 in mediating SNN formation. C_LIO_LIQM/MM MD metadynamics simulations support experimental findings. C_LI

biochemistry↗

bioRxiv2025

Understanding the biochemical significance of the mitochondrial pyruvate carrier in the rodent malarial parasite, Plasmodium berghei

Mitochondrial pyruvate carrier (MPC) protein has been established as the key pyruvate transporter across the inner mitochondrial membrane. In Plasmodium, flux through the tricarboxylic acid (TCA) cycle is low in the asexual stages that is upregulated in the gametocytes with greater utilization of glutamine than glucose to drive the cycle. Acetyl-CoA in the mitochondrion has been established to be essential for the parasite. In this context, we established the biochemical function of P. berghei MPC as a pyruvate transporter through functional complementation in yeast. In P. berghei, both subunits, MPC1 and MPC2 were found to localize to the mitochondrion and surprisingly, were found to be non-essential for the blood stages and for ookinete formation. Through metabolite tracing studies using 13C6-glucose and 13C515N2 L-glutamine, we have derived the biochemical basis for this observed phenotype of {Delta}mpc1{Delta}mpc2 parasites. Our studies suggest the presence of an aminotransferase localized to the mitochondrion that converts alanine to pyruvate, leading to the continued production of acetyl-CoA in the organelle even in the absence of MPC. This highlights the existence of metabolic plasticity, a survival strategy, that enables the utilization of different metabolic precursors for the generation of acetyl-CoA in the mitochondrion.

biochemistry↗

bioRxiv2023

Enantioselectivity in the Enzymatic Dehydration of Malate and Tartrate: Mirror Image Specificities of Structurally Similar Dehydratases

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 anti- elimination 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 rationalised 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.

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↗

bioRxiv2022

Mechanistic insights into the functioning of GMP synthetase: a two-subunit, allosterically regulated, ammonia tunnelling enzyme

Guanosine 5-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5-monophosphate (XMP) to GMP are comprised of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase and the ammonia thus generated is tunnelled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia tunnelling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channelling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure and cross-linking mass spectrometry guided integrative modelling. pH dependence of enzyme kinetics establish that ammonia is tunnelled across the subunits with the lifetime of the complex being [≤] 0.5 s. The crystal structure of XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/481963v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1eb7261org.highwire.dtl.DTLVardef@a25d02org.highwire.dtl.DTLVardef@1885ed7org.highwire.dtl.DTLVardef@ab189_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Structural basis for the hyperthermostability of an archaeal glutaminase induced by post-translational succinimide formation

Stability of proteins from hyperthermophiles enabled by reduction of conformational flexibility is realized through various mechanisms. Presence of a stable, hydrolysis-resistant succinimide arising from cyclization of the side chains of aspartyl/asparaginyl residues with backbone amide -NH of the succeeding residue would restrain the torsion angle {Psi}. Here, we describe the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase (MjGATase) and address the mechanism of a succinimide-induced increased thermostability using molecular dynamics simulations. This study reveals the interplay of negatively charged electrostatic shield and n[->]{pi}* interactions in preventing succinimide hydrolysis. The stable succinimidyl residue induces formation of a conformational-lock, reducing protein flexibility. Protein destabilization upon replacement with the {Phi}-restricted prolyl residue highlights the specificity of the conformationally restrained succinimidyl residue in imparting hyperthermostability. The conservation of succinimide-forming tripeptide sequence (E(N/D)(E/D)) in a group of archaeal GATases suggests an adaptation of this otherwise detrimental post-translational modification as an inducer of thermostability.

biochemistry↗

bioRxiv2020

Leptospira interrogans triosephosphate isomerase: exploring the structural determinants of stability, high reaction rate and specificity.

Leptospires are zoonotic pathogens that cause significant socio-economic burden in developing countries, world-wide. The pathogenic species Leptospira interrogans (Li) is an important and interesting target for investigating the enzymes essential to its metabolic needs and adaptations. We cloned and expressed triosephosphate isomerase (LiTIM), a central metabolic flux regulator of Li, in AA200, E. coli TIM null strain. LiTIM was obtained as an active dimer (D-GAP[->]DHAP kcat = 1740 s-1 and Km (D-GAP) = 0.21 mM, at 25 {degrees}C) with mid-transition concentrations, Cm, 0.8 mM and 2.6 mM, respectively, for guanidine hydrochloride and urea induced equilibrium unfolding. We report the high resolution X-ray structures of LiTIM in apo and substrate (DHAP) bound forms. Our analysis highlights key features of TIM that regulate the mode of substrate binding and transition state stabilization and thus play a decisive role in attainment of high proficiency for the isomerisation reaction while avoiding the elimination reaction. Unexpected differences in the effect of temperature on stability and activity were observed for the three mesophilic pathogenic TIMs viz. from Li, Plasmodium falciparum (Pf) and Trypanosoma brucei (Tb). LiTIM and TbTIM (Tm = 46.5 {degrees}C) were more susceptible to unfolding and precipitation compared to PfTIM (Tm = 67 {degrees}C). In contrast, the initial (or zero point) activity of PfTIM rises till 50 {degrees}C and saturates unlike LiTIM and TbTIM which show a rise till 55 {degrees}C and 60 {degrees}C, respectively. These observations could be rationalized by sequence comparison and examination of the structures of the three TIMs.

biochemistry↗

bioRxiv2019

Interactions of helices on interdomain interface couple the chemistry of intermediate formation with allostery in Plasmodium falciparum GMP synthetase

GMP synthetase catalyzes the substitution of the C2 oxo-group of the purine base in XMP with an amino-group generating GMP, the last step in the biosynthesis of GMP. This reaction involves a series of catalytic events that include hydrolysis of Gln generating ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. Inherent to the functioning of GMP synthetases is bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain by substrates binding to the ATPPase domain, synchronization of the two catalytic events and tunnelling of ammonia from the GATase to the ATPPase domain. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and, steady-state and transient kinetic assays on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. The results map the residues critical for catalysis in the ATPPase domain to the helices 11 and 12 that are located at the interdomain interface, and the lid-loop that follows 11. This apart, perturbing interdomain interactions involving residues on 11 and 12 impairs GATase activation. These results imply that this arrangement of helices at the domain interface with residues that play roles in ATPPase catalysis as well as domain crosstalk enables coupling ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

biochemistry↗

bioRxiv2018

Metabolic proof-reading in Plasmodium berghei: essentiality of phosphoglycolate phosphatase

Plasmodium falciparum (Pf)4-nitrophenylphosphatase was earlier shown to be involved in vitamin B1 metabolism by Knockel et al., (Mol. Biochem. Parasitol. 2008, 157, 241-243). An independent BLASTp search showed that the protein had significant homology with phos-phoglycolate phosphatase from mouse, human and yeast, and prompted us to re-investigate the biochemical properties of the recombinant Plasmodium enzyme. Owing to the insoluble nature of the Pf enzyme, an extended substrate screen and biochemical characterization was performed on its P. berghei (Pb) homolog that led to the identification of 2-phosphoglycolate and 2-phospho L-lactate as the relevant physiological substrates. 2-phosphoglycolate is known to be generated during repair of damaged DNA ends whereas, 2-phospho L-lactate is a product of pyruvate kinase side reaction. These metabolites are potent inhibitors of the key glycolytic enzymes, triosephosphate isomerase and phosphofructokinase, and hence clearance of these toxic metabolites is vital for cell survival and functioning. Gene knockout studies conducted in P. berghei revealed the essential nature of this conserved metabolic proof-reading enzyme.

biochemistry↗

bioRxiv2018

PBiochemical and physiological investigations on adenosine 5’ monophosphate deaminase from Plasmodium spp

AbbreviationsHGXPRT - Hypoxanthine-guanine-xanthine phosphoribosyltransferase, ADSS - Adenylosuccinate synthetase, ASL - Adenylosuccinate lyase, GMPS - Guonosine monophosphate synthetase, IMPDH - Inosine monophosphate dehydrogenase, ISN1 - Inosine monophosphate specific nucleotidase, PNP - Purine nucleoside phosphorylase\n\nSummaryInterplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter-conversion of ADP, ATP and AMP, plays a major role in maintaining AEC, and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in-turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be non-essential for intra-erythrocytic growth of the knockout parasites. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over-expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti-parasitic agents.

biochemistry↗

bioRxiv2018

Visualizing Deep Mutational Scan Data

Site-directed and random mutagenesis are biochemical tools to obtain insights into the structure and function of proteins. Recent advances such as deep mutational scan have allowed a complete scan of all the amino acid positions in a protein with each of the 19 possible alternatives. Mapping out the phenotypic consequences of thousands of single point mutations in the same protein is now possible. Visualizing and analysing the rich data offers an opportunity to learn more about the effects of mutations, for a better understanding and engineering of proteins. This work focuses on such visualization analyses applied to the mutational data of TEM-1 {beta}-lactamase. The data is examined in the light of the expected biochemical effects of single point mutations, with the goal of reinforcing or retraining the intuitions. Individual attributes of the amino acid mutations such as the solvent accessible area, charge type change, and distance from the catalytic center capture most of the relevant functional effects. Visualizing the data suggests how combinations of these attributes can be used for a better classification of the effects of mutations, when independently they do not offer a high predictability.

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↗