RESUMO
Xyloglucan endotransglycosylase/hydrolases (XTHs) are cell wall enzymes with hydrolase (XEH) and/or endotransglycosylase (XET) activities. As they are involved in the modification of the xyloglucans, a type of hemicellulose present in the cell wall, they are believed to be very important in different processes, including growth, development, and fruit ripening. Previous studies suggest that XTHs might play a key role in development and ripening of Fragaria chiloensis fruit, and its characterization is pending. Therefore, in order to provide a biochemical characterization of the FcXTH2 enzyme to explain its possible role in strawberry development, the molecular cloning and the heterologous expression of FcXTH2 were performed. The recombinant FcXTH2 was active and displayed mainly XEH activity. The optimal pH and temperature are 5.5 and 37 °C, respectively. A KM value of 0.029 mg mL-1 was determined. Additionally, its protein structural model was built through comparative modeling methodology. The model showed a typically ß-jelly-roll type folding in which the catalytic motif was oriented towards the FcXTH2 central cavity. Using molecular docking, protein-ligand interactions were explored, finding better interaction with xyloglucan than with cellulose. The data provided groundwork for understanding, at a molecular level, the enzymatic mechanism of FcXTH2, an important enzyme acting during the development of the Chilean strawberry.
Assuntos
Fragaria/enzimologia , Frutas/enzimologia , Glicosiltransferases/metabolismo , Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Chile , Clonagem Molecular , Fragaria/genética , Fragaria/crescimento & desenvolvimento , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucanos/química , Glucanos/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Concentração de Íons de Hidrogênio , Hidrolases/química , Hidrolases/genética , Cinética , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Domínios Proteicos , Temperatura , Xilanos/química , Xilanos/metabolismoRESUMO
Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies confirmed XET activity for PrXTH1 protein from radiata pine. XTHs could interact with many hemicellulose substrates, but the favorite substrate of PrXTH1 is still unknown. The prediction of union type and energy stability of the complexes formed between PrXTH1 and different substrates (XXXGXXXG, XXFGXXFG, XLFGXLFG and cellulose) were determined using bioinformatics tools. Molecular Docking, Molecular Dynamics, MM-GBSA and Electrostatic Potential Calculations were employed to predict the binding modes, free energies of interaction and the distribution of electrostatic charge. The results suggest that the enzyme formed more stable complexes with hemicellulose substrates than cellulose, and the best ligand was the xyloglucan XLFGXLFG (free energy of -58.83⯱â¯0.8â¯kcalâ¯mol-1). During molecular dynamics trajectories, hemicellulose fibers showed greater stability than cellulose. Aditionally, the kinetic properties of PrXTH1 enzyme were determined. The recombinant protein was active and showed an optimal pH 5.0 and optimal temperature of 37⯰C. A Km value of 20.9â¯mM was determined for xyloglucan oligomer. PrXTH1 is able to interact with different xyloglycans structures but no activity was observed for cellulose as substrate, remodeling cell wall structure in response to inclination.
Assuntos
Glicosiltransferases/metabolismo , Pichia/fisiologia , Proteínas de Plantas/metabolismo , Parede Celular/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas/fisiologia , Glicosiltransferases/fisiologia , Cinética , Simulação de Acoplamento Molecular , Pichia/enzimologia , Pichia/metabolismo , Proteínas de Plantas/fisiologia , Proteínas Recombinantes , Especificidade por SubstratoRESUMO
Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies suggest that XTHs might play a key role in ripening of Fragaria chiloensis fruit as FcXTH1 transcripts increase as fruit softens. FcXTH1 protein sequence contains a conserved N-glycosylation site adjacent to catalytic residues. The FcXTH1 structure was built through comparative modeling methodology, the structure displays a ß-jellyroll-type folding with a curvature generated by eight antiparallel ß-sheets that holds the catalytic motif that is oriented towards the central cavity of the protein. Through Molecular Dynamic Simulations (MDS) analyses the protein-ligand interactions of FcXTH1 were explored, finding a better interaction with xyloglucans than cellulose. Nevertheless, the stability of the protein-ligand complex depends on the glycosylation state of FcXTH1: better energy interactions were determined for the glycosylated protein. As a complement, the molecular cloning and heterologous expression of FcXTH1 in Pichia pastoris was performed, and the recombinant protein was active and displayed strict XET activity. A KM value of 17.0 µM was determined for xyloglucan oligomer. The deglycosylation of FcXTH1 by PNGase-F treatment affects its biochemical properties (increase KM and reduce kcat/KM ratio) and reduces its stability. As a conclusion, glycosylation of FcXTH1 is important for its biological function.