Luis Morales-Quintana performed a PhD in Science with mention in plant genetic engineering from the University of Talca and received the degree at 2013. Now work as young professor at the Institute of Biological Sciences of University of Talca, and is part of the Funtional genomics, Biochemistry and Plant physiology group that is lead by Dr. Raúl Herrera.His research main topic focuses in the study of molecular aspects in the formation and degradation of plant cell wall. The strategies used involve: structural bioinformatics, biochemical characterization, kinetics studies, and functional genomics. To describe genes, proteins, enzymes and how they modulate the response to different events occurring in the plant cell wall.
Strawberry fruit has a very short shelf life due to its fast softening rate. Fruit softening has been shown to be related to cell wall degradation. As changes in the hemicellulosic fraction have been reported during ripening of Fragaria chiloensis fruit, the participation of xyloglucan endotransglycosilase/hydrolase (XTH) was studied. In previous studies FcXTH1 gene was identified in F. chiloensis fruit with high homology to other plant XTHs. FcXTH1 protein sequence contains a conserved N-glycosylation site adjacent to predicted catalytic residues. To gain insight about the mechanism of action of FcXTH1 enzyme at the molecular level, the 3D structure of FcXTH1 was built through comparative modeling methodology. The model obtained displays a β-jellyroll–type structure that comprises one α-helix, three 310 helices and fifteen β-sheets; a curvature generated by eight antiparallel β-sheets holds the catalytic DEIDFEFLG motif that is oriented towards the central cavity of the protein. MM-GBSA analyses explored the interaction of FcXTH1 protein and a set of putative substrates, 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 in the glycosylated protein. To corroborate this, the molecular cloning and heterologous expression of FcXTH1 in Pichia pastoris was performed, the recombinant protein was active and displayed XET activity. The activities were stable at 4ºC (> 50% activity remaining after 5 days). A KM value of 16.9 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.
Research supported by CONICYT/PAI 79140027, Anillo ACT-1110 and Fondecyt 11150543 projects