Two-dimensional (2D) d-Silicates from abundant natural minerals

In the last decade, the materials community has been exploring new 2D materials (graphene, metallene, TMDs, TMCs, MXene, among others) that have unique physical and chemical properties. Recently, a new family of 2D materials, the so-called 2D silicates, have been proposed. They are predicted to exhibit exciting properties (such as high catalytic activity, piezoelectricity, and 2D magnetism). In the current work, we demonstrate a generic approach to the synthesis of large-scale 2D silicates from selected minerals, such as Diopside (d). Different experimental techniques were used to confirm the existence of the 2D structures (named 2D-d-silicates). DFT simulations were also used to gain insight into the structural features and energy harvesting mechanisms (flexoelectric response generating voltage up to 10 V). The current approach is completely general and can be utilized for large-scale synthesis of 2D silicates and their derivatives, whose large-scale syntheses have been elusive

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from <em>Debaryomyces hansenii</em>

<div><p>Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from <em>Debaryomyces hansenii</em> (<em>Dh</em>XR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. <em>Dh</em>XR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-<em>Dh</em>XR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-<em>Dh</em>XR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-<em>Dh</em>XR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.</p> </div

Molecular Basis of Peptide Recognition in Metallopeptidase Dug1p from <i>Saccharomyces cerevisiae</i>

Dug1p, a M20 family metallopeptidase and human orthologue of carnosinase, hydrolyzes Cys-Gly dipeptide, the last step of glutathione (GSH) degradation in <i>Saccharomyces cerevisiae</i>. Molecular bases of peptide recognition by Dug1p and other M20 family peptidases remain unclear in the absence of structural information about enzyme–peptide complexes. We report the crystal structure of Dug1p at 2.55 Å resolution in complex with a Gly-Cys dipeptide and two Zn²⁺ ions. The dipeptide is trapped in the tunnel-like active site; its C-terminus is held by residues at the S1′ binding pocket, whereas the S1 pocket coordinates Zn²⁺ ions and the N-terminus of the peptide. Superposition with the carnosinase structure shows that peptide mimics the inhibitor bestatin, but active site features are altered upon peptide binding. The space occupied by the N-terminus of bestatin is left unoccupied in the Dug1p structure, suggesting that tripeptides could bind. Modeling of tripeptides into the Dug1p active site showed tripeptides fit well. Guided by the structure and modeling, we examined the ability of Dug1p to hydrolyze tripeptides, and results show that Dug1p hydrolyzes tripeptides selectively. Point mutations of catalytic residues do not abolish the peptide binding but abolish the hydrolytic activity, suggesting a noncooperative mode in peptide recognition. In summary, results reveal that peptides are recognized primarily through their amino and carboxyl termini, but hydrolysis depends on the properties of peptide substrates, dictated by their respective sequences. Structural similarity between the Dug1p–peptide complex and the bestatin-bound complex of CN2 suggests that the Dug1p–peptide structure can be used as a template for designing natural peptide inhibitors

Steady state kinetic parameters for different carbonyl substrates catalyzed by <i>Dh</i>XR and kinetic analyses of D-xylose reduction by <i>Dh</i>XR in the presence of non-xylose sugars.

<p>Note: G, Galactose; A, Arabinose; R, Rhamnose.</p

Steady-state kinetic characterization of <i>Dh</i>XR.

<p>Substrate specificity of <i>Dh</i>XR checked and kinetic data were fit to Michaelis-Menten model as described in methods; enzyme concentration is same for all experiments (0.18 µM). A) Kinetic study using D-xylose as substrate; B) Comparative kinetic study using different carbonyl substrates; C) Examination of <i>Dh</i>XR kinetic properties towards D-xylose in the presence of fixed amounts (40 mM) of non-xylose substrates.</p

Structural analyses of L-rhamnose interaction with apoenzyme.

<p>A) F_o−F_c omit-electron density map (2.5 σ level) shows L-rhamnose backbones and hydroxyl groups and 2 F_o−F_c electron density map (1.0 σ level) also shows rhamnose is bound to active site cavity. B) Interactions of bound L-rhamnose with side chains of residues lining the active site cavity. The aldehyde part of ligand is aligned towards side chain of Y217 with O1 forming strong hydrogen bond with OH of Y217 and also interacting with near by aromatic side chains. C) Superposition of NADPH bound structure to apoenzyme-rhamnose complex. The plane of side chain of Y217 tilted nearly perpendicularly in cofactor bound structure, causing to be atop of NADPH. Apoenzyme-rhamnose complex is shown in green and NADPH bound complex is shown in blue.</p

Specific activities of <i>Dh</i>XR with xylose in presence of different carbonyl substrates.

*<p>amount of substrate added in 100 mM of D-xylose reaction.</p

Determination of equilibrium binding constants for ligands binding to active site mutants.

*<p>all units are in mM.</p

Fluorescence quenching titrations of <i>Dh</i>XR mutants with different substrates.

<p>H109A (□); K76A (•); Y47A (○); Protein concentration was 2.8×10⁻⁷ M; the data from both titrations were fit to two non-identical site model (eq 1); A) ribose; B) D-arabinose; C) galactose; D) Sucrose; The solid line represents the best fit to the data.</p