Resveratrol glucosylation by GTF-SI from Streptococcus mutans: computational insights into a GH70 family enzyme.

Resveratrol, a polyphenolic compound known for its health benefits but limited by poor water solubility and low bioavailability, represents a valuable substrate for glucosylation by carbohydrate-active enzymes such as glucosyltransferase-SI (GTF-SI). Using quantum mechanics/molecular mechanics (QM/MM) calculations and molecular dynamics simulations, this study reveals the atomic scale dynamics of resveratrol glucosylation by wild-type GTF-SI. This enzyme exhibited an energy barrier of 8.8 kcal mol-1 and an exothermic process, both consistent with experimental data of similar enzymes. We report a concerted and synchronous reaction mechanism for the catalytic step, characterized by an oxocarbenium ion-like transition state, and elucidate a conformational itinerary of the glucosyl moiety (4H3/E3) → [E3]‡ → 4C1, which aligns with the consistent patterns observed across enzymes of the GH13 and GH70 families. A key interaction was observed between Asp477 and the OH group on carbon 6 of the glucosyl moiety, together with a 2.0 kcal mol-1 transition state stabilization by three water molecules within the active site. Comparative insights with the previously studied Q345F SP enzyme system shed light on the unique and common features that govern transglucosylation reactions. Importantly, the calculated activation barriers strongly support the capability of GTF-SI to facilitate resveratrol glucosylation. This study advances our understanding of the transglucosylation reaction and opens up new ways for the glycodiversification of organic compounds such as polyphenols, thus expanding their potential applications in the food, cosmetic, and pharmaceutical industries.

Insight into the Role of Active Site Protonation States and Water Molecules in the Catalytic Inhibition of DPP4 by Vildagliptin.

Vildagliptin (VIL) is an antidiabetic drug that inhibits dipeptidyl peptidase-4 (DPP4) through a covalent mechanism. The molecular bases for this inhibitory process have been addressed experimentally and computationally. Nevertheless, relevant issues remain unknown such as the roles of active site protonation states and conserved water molecules nearby the catalytic center. In this work, molecular dynamics simulations were applied to examine the structures of 12 noncovalent VIL-DPP4 complexes encompassing all possible protonation states of three noncatalytic residues (His126, Asp663, Asp709) that were inconclusively predicted by different computational tools. A catalytically competent complex structure was only achieved in the system with His126 in its ε-form and nonconventional neutral states for Asp663/Asp709. This complex suggested the involvement of one water molecule in the catalytic process of His740/Ser630 activation, which was confirmed by QM/MM simulations. Our findings support the suitability of a novel water-mediated mechanism in which His740/Ser630 activation occurs concertedly with the nucleophilic attack on VIL and the imidate protonation by Tyr547. Then, the restoration of His740/ Tyr547 protonation states occurs via a two-water hydrogen bonding network in a low-barrier process, thus describing the final step of the catalytic cycle for the first time. Additionally, two hydrolytic mechanisms were proposed based on the hydrogen bonding networks formed by water molecules and the catalytic residues along the inhibitory mechanism. These findings are valuable to unveil the molecular features of the covalent inhibition of DPP4 by VIL and support the future development of novel derivatives with improved structural or mechanistic profiles.

Elucidation of the Reaction Mechanism of Cavia porcellus l-Asparaginase: A QM/MM Study.

The l-asparaginase (l-ASNase) enzyme catalyzes the conversion of the non-essential amino acid l-asparagine into l-aspartic acid and ammonia. Importantly, the l-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species (Escherichia coli and Erwinia chrysanthemi). Therefore, one way to solve these side effects is the use of l-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus l-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus l-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of l-asparagine into l-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the rate-limiting step of the reaction. In this step, the nucleophile (Thr19) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol-1, at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol-1 (derived from the kcat value of 38.6 s-1). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.

Glucosylation mechanism of resveratrol through the mutant Q345F sucrose phosphorylase from the organism Bifidobacterium adolescentis: a computational study.

Mainly due to their great antioxidant, anti-inflammatory and anticancer capacities, natural polyphenolic compounds have many properties with important applications in the food, cosmetic and pharmaceutical industries. Unfortunately, these molecules have very low water solubility and bioavailability. Glucosylation of polyphenols is an excellent alternative to overcome these drawbacks. Specifically, for the natural polyphenol resveratrol this process is very inefficiently performed by the native enzyme sucrose phosphorylase (BaSP) from the organism Bifidobacterium adolescentis (4%). However, the Q345F point mutation of the sucrose phosphorylase (BaSP Q345F) has been shown to achieve 97% monoglucosylation for the same substrate and the mechanism is still unknown. This report presents an analysis of MD simulations performed with the BaSP Q345F and BaSP systems in complex with resveratrol monoglucoside, followed by a study of the transglucosylation reaction of the mutant enzyme BaSP Q345F with resveratrol through the QM/MM hybrid method. With respect to the MD simulations, both protein structures showed greater similarity to the phosphate-binding conformation, and a larger active site and conformational changes in certain structures were found for the mutant system compared with the native enzyme; all this is in agreement with experimental data. With regard to the QM/MM calculations, the structure of an oxocarbenium ion-like transition state and the minimum energy adiabatic path (MEP) that connects the reactants with the products were obtained with a 20.3 kcal mol-1 energy barrier, which fits within the known experimental range for this type of enzyme. Finally, the analyses performed along the MEP suggest a concerted but asynchronous mechanism. In particular, they show that the interactions involving the residues of the catalytic triad (Asp192, Glu232, and Asp290) together with two water molecules at the active site strongly contribute to the stabilization of the transition state. The understanding of this glucosylation mechanism of the polyphenol resveratrol carried out by the mutant sucrose phosphorylase enzyme presented in this work could serve as the basis for subsequent studies on related carbohydrate-active enzymes.

Molecular modeling study on the differential microtubule-stabilizing effect in singly- and doubly-bonded complexes with peloruside A and paclitaxel.

Microtubules (MT) are dynamic cytoskeletal components that play a crucial role in cell division. Disrupting MT dynamics by MT stabilizers is a widely employed strategy to control cell proliferation in cancer therapy. Most MT stabilizers bind to the taxol (TX) site located at the luminal interface between protofilaments, except laulimalide and peloruside A (PLA), which bind to an interfacial pocket on outer MT surface. Cryo-electron microscopy MTs reconstructions have shown differential structural effects on the MT lattice in singly- and doubly-bonded complexes with PLA, TX, and PLA/TX, as PLA is able to revert the lattice heterogeneity induced by TX association leading to more regular MT assemblies. In this work, fully-atomistic molecular dynamics simulations were employed to examine the single and double association of MT stabilizers to reduced MT models in the search for structural and energetic evidence that could be related to the differential regularization and stabilization effects exerted by PLA and TX on the MT lattice. Our results revealed that the double association of PLA/TX (a) strengthens the lateral contact between tubulin dimers compared to singly-bonded complexes, (b) favors a more parallel arrangement between tubulin dimers, and (c) induces a larger restriction in the interdimeric conformational motion increasing the probability of finding structures consistent with 13-protofilaments arrangements. These results and are valuable to increase understanding about the molecular mechanism of action of MT stabilizers, and could account for an overstabilization of MTs in doubly-bonded complexes compared to singly-bonded systems.

Structural insight into the role of Gln293Met mutation on the Peloruside A/Laulimalide association with αß-tubulin from molecular dynamics simulations, binding free energy calculations and weak interactions analysis.

Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of ß-tubulin and promote microtubule stabilization by bridging two adjacent αß-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique ß-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αß-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.