RESUMO
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) â [E3] â 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.
Assuntos
Glucosiltransferases , Streptococcus mutans , Humanos , Resveratrol , Glucosiltransferases/química , Simulação de Dinâmica Molecular , ÁguaRESUMO
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.
Assuntos
Bifidobacterium adolescentis , Domínio Catalítico , Glucosiltransferases , Polifenóis , Resveratrol , ÁguaRESUMO
Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl-thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP(-)). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP(-) has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP(-)) and a second pyruvate molecule. Our results suggest that additional acid-base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid-base properties of the HEThDP(-) intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP(-) intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle.