@article {121924, title = {Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin.}, journal = {Biochemistry}, year = {2023}, month = {2023 Jan 03}, abstract = {

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. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and 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 density functional theory (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.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.2c00541}, author = {Bellur, Asutosh and Das, Soumik and Jayaraman, Vijay and Behera, Sudarshan and Suryavanshi, Arpitha and Balasubramanian, Sundaram and Balaram, Padmanabhan and Jindal, Garima and Balaram, Hemalatha} } @article {117960, title = {Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme}, journal = {BIOCHEMISTRY}, volume = {61}, year = {2022}, month = {09/2022}, pages = {1988-2006}, abstract = {

Guanosine 5 {\textquoteright}-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5 {\textquoteright}-mono-phosphate (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. In ammonia channeling 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 channeling 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 modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being \<= 0.5 s. The crystal structure of the 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.

}, doi = {10.1021/acs.biochem.2c001511988}, author = {Shivakumaraswamy, Santosh and Kumar, Sanjeev and Bellur, Asutosh and Polisetty, Satya Dev and Balaram, Hemalatha} } @article {96416, title = {Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation.}, journal = {Biophys J}, year = {2021}, month = {2021 Jul 21}, abstract = {

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. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase (MjGATase) and using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100 C, imparted by an unexpectedly stable succinimidyl residue (SNN) at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side chain carboxylate group of its succeeding residue Asp110 as well as through n{\textrightarrow}π interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2021.07.014}, author = {Dongre, Aparna Vilas and Das, Sudip and Bellur, Asutosh and Kumar, Sanjeev and Chandrashekarmath, Anusha and Karmakar, Tarak and Balaram, Padmanabhan and Balasubramanian, Sundaram and Balaram, Hemalatha} } @article {1015, title = {Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine-Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides.}, journal = {Chemphyschem}, volume = {16}, year = {2015}, month = {2015 Jul 20}, pages = {2172-81}, abstract = {

Plasmodium falciparum (Pf) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9-deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs. Through the use of these probes it is found that non-covalent interactions stabilise the pre-transition state of the HGPRT-catalysed reaction. Vibrational spectra reveal that the bound substrates are extensively distorted, the carbonyl bond of nucleobase moiety is weakened and the substrate is destabilised along the reaction coordinate. Raman shifts of the human and Pf enzymes are used to quantify the differing degrees of hydrogen bonding in the homologues. A decreased Raman cross-section in enzyme-bound 9-deazaguanine (9DAG) shows that the phenylalanine residue (Phe186 in human and Phe197 in Pf) of HGPRT stacks with the nucleobase. Differential loss of the Raman cross-section suggests that the active site is more compact in human HGPRT as compared to the Pf enzyme, and is more so in the phosphoribosyl pyrophosphate (PRPP) complex 9DAG-PRPP-HGPRT than in 9-deazahypoxanthine (9DAH)-PRPP-HGPRT.

}, issn = {1439-7641}, doi = {10.1002/cphc.201500084}, author = {Karnawat, Vishakha and Gogia, Spriha and Balaram, Hemalatha and Puranik, Mrinalini} }