Metabolic Flexibility and Essentiality of the Tricarboxylic Acid Cycle in .

The complete tricarboxylic acid (TCA) cycle, comprising a series of 8 oxidative reactions, occurs in most eukaryotes in the mitochondria and in many prokaryotes. The net outcome of these 8 chemical reactions is the release of the reduced electron carriers NADH and FADH, water, and carbon dioxide. The parasites of the .

Amino Acid Chirality: Stereospecific Conversion and Physiological Implications.

The absolute configuration dictates the biological role of chiral molecules in the living world. This is best exemplified by all ribosomally synthesized polypeptides having chiral amino acids only in the l-configuration. However, d-amino acids are also associated with various vital biological processes such as peptidoglycan of the bacterial cell wall, ligands for neurotransmitters, molecules involved in signaling, and precursors of metabolites, to name a few.

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 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.

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, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which 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 (Mj) as a model along with comparative analysis with the only other available class-I FH structure from (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes.

Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme.

Guanosine 5'-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5'-monophosphate (XMP) to GMP, are composed 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 an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP.

Tertiary and Quaternary Structure Organization in GMP Synthetases: Implications for Catalysis.

Glutamine amidotransferases, enzymes that transfer nitrogen from Gln to various cellular metabolites, are modular, with the amidotransferase (GATase) domain hydrolyzing Gln, generating ammonia and the acceptor domain catalyzing the addition of nitrogen onto its cognate substrate. GMP synthetase (GMPS), an enzyme in the de novo purine nucleotide biosynthetic pathway, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP. The reaction involves activation of XMP though adenylation by ATP in the ATP pyrophosphatase (ATPPase) active site, followed by channeling and attack of NH generated in the GATase pocket.

Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation.

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue.

Toward Developing Intuitive Rules for Protein Variant Effect Prediction Using Deep Mutational Scanning Data.

Protein structure and function can be severely altered by even a single amino acid mutation. Predictions of mutational effects using extensive artificial intelligence (AI)-based models, although accurate, remain as enigmatic as the experimental observations in terms of improving intuitions about the contributions of various factors. Inspired by Lipinski's rules for drug-likeness, we devise simple thresholding criteria on five different descriptors such as conservation, which have so far been limited to qualitative interpretations such as high conservation implies high mutational effect.

Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase.

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate 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. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk.

How a purine salvage enzyme singles out the right base.

Two phosphoribosyltransferases in the purine salvage pathway exhibit exquisite substrate specificity despite the chemical similarity of their distinct substrates, but the basis for this discrimination was not fully understood. Ozeir now employ a complementary biochemical, structural, and computational approach to deduce the chemical constraints governing binding and propose a distinct mechanism for catalysis in one of these enzymes, adenine phosphoribosyltransferase. These insights, built on data from an unexpected finding, finally provide direct answers to key questions regarding these enzymes and substrate recognition more generally.

Biochemical and physiological investigations on adenosine 5' monophosphate deaminase from Plasmodium spp.

The interplay 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.

Phosphoglycolate phosphatase is a metabolic proofreading enzyme essential for cellular function in .

(Pf) 4-nitrophenylphosphatase has been shown previously to be involved in vitamin B1 metabolism. Here, conducting a BLASTp search, we found that 4-nitrophenylphosphatase from Pf has significant homology with phosphoglycolate phosphatase (PGP) from mouse, human, and yeast, prompting us to reinvestigate the biochemical properties of the enzyme. Because the recombinant PfPGP enzyme is insoluble, we performed an extended substrate screen and extensive biochemical characterization of the recombinantly expressed and purified homolog from (Pb), leading to the identification of 2-phosphoglycolate and 2-phospho-L-lactate as the relevant physiological substrates of PbPGP.

Biochemical characterization and essentiality of fumarate hydratase.

(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 from Fumarate is generated in large quantities in the parasite as a by-product 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 , but biochemical characterization of PfFH that may provide leads for the development of specific inhibitors is lacking.

Probing the influence of non-covalent contact networks identified by charge density analysis on the oxidoreductase BacC.

Bacillus subtilis BacC is an oxidoreductase involved in the biosynthesis of the potent antibiotic bacilysin. The crystal structure of BacC was determined at 1.19 Å resolution.

Role of W181 in modulating kinetic properties of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

Hypoxanthine-guanine-xanthine phosphoribosyltransference (HGXPRT), a key enzyme in the purine salvage pathway of the malarial parasite, Plasmodium falciparum (Pf), catalyses the conversion of hypoxanthine, guanine, and xanthine to their corresponding mononucleotides; IMP, GMP, and XMP, respectively. Out of the five active site loops (I, II, III, III', and IV) in PfHGXPRT, loop III' facilitates the closure of the hood over the core domain which is the penultimate step during enzymatic catalysis. PfHGXPRT mutants were constructed wherein Trp 181 in loop III' was substituted with Ser, Thr, Tyr, and Phe.

Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase.

Protein ageing is often mediated by the formation of succinimide intermediates. These short-lived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into β-aspartyl or D-aspartyl residues. Here we report the presence of a highly stable succinimide intermediate in the glutaminase subunit of GMP synthetase from the hyperthermophile Methanocaldoccocus jannaschii.

Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase.

Atomistic molecular dynamics (MD) simulations coupled with the metadynamics technique were carried out to delineate the product (PPi.2Mg and IMP) release mechanisms from the active site of both human (Hs) and Plasmodium falciparum (Pf) hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT). An early movement of PPi.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction.

Kinetic mechanism of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) exhibits a kinetic mechanism that differs from that of the human homolog. Human HGPRT follows a steady-state ordered mechanism, wherein PRPP binding precedes the binding of hypoxanthine/guanine and release of product IMP/GMP is the rate limiting step. In the current study, initial velocity kinetics with PfHGXPRT indicates a steady-state ordered mechanism, wherein xanthine binding is conditional to the binding of PRPP.

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective.

Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation.

GMP synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear. Crystal structures of Plasmodium falciparum GMPS in conjunction with mutational and enzyme kinetic studies reported here provide evidence that an 85° rotation of the GATase domain is required for ammonia channelling and thus for the catalytic activity of this two-domain enzyme. We suggest that conformational changes in helix 371-375 holding catalytic residues and in loop 376-401 along the rotation trajectory trigger the different steps of catalysis, and establish the central role of Glu374 in allostery and inter-domain crosstalk.

Structural and dynamical correlations in PfHGXPRT oligomers: A molecular dynamics simulation study.

PfHGXPRT is a key enzyme involved in purine nucleotide salvage pathway of the malarial parasite, Plasmodium falciparum. Atomistic molecular dynamics simulations have been performed on two types of PfHGXPRT dimers (D1 and D3) and its tetramer in their apo and ligand-bound states. A significant event in the catalytic cycle is the dynamics of a gate that provides access for the ligand molecules to the reaction center.