Assessment of XXT1 and XXT5 Modes of Substrate Binding and Structure‐Solubility Relationship Using Site‐Directed Mutagenesis
Authors: Ehrlich Jacqueline J , Culbertson Alan , Zabotina Olga 
Published: June 2021

Plant cell wall polysaccharides are the largest source of biopolymers on Earth and have numerous industrial applications such as food, fiber, biofuels and biomaterials. However, development is impeded because of the dearth of structural information for plant glycosyltransferases, the key enzymes responsible for polysaccharide biosynthesis. A set of specific transmembrane proteins synthesizes the most abundant hemicellulosic polysaccharide, xyloglucan. Two Xyloglucan Xylosyltransferases 1 and 5 (XXT1 and XXT5, respectively) add a xylose moiety to the glucan backbone, as the initial step in xyloglucan branching. The structure of XXT1 was solved using X‐ray crystallography and key information about residues involved in catalysis and protein solubility was obtained through site‐directed mutagenesis, size exclusion chromatography (SEC), and activity assays using a newly developed PKLD enzyme assay.In order to reveal modes of substrate binding and ascertain the conclusions drawn from the crystal structure of XXT1, site directed mutagenesis was performed to assess the critical amino acids involved in glycosyltransferase activity. The mutation of the residues involved in coordination of Mn2+ such as Asp229Ala, Asp227Ala/Asp229Ala, and His337Ala, significantly reduced enzyme activity. Mutations Lys382Ala, Asp317Ala, Asp318Ala, and Gln319Ala reduced activity most due to their importance in hydrogen bonding. Whereas, Ser228Ala and Asn268Ala mutants did not show changes in activity.The next objective of our study was to investigate the suitability of XXT5 for crystallization. The apparent lower solubility of XXT5 compared with XXT1 prevents the former protein to be crystallized. First, a homology model of XXT5 has been generated from the crystal structure of XXT1 and differences in surface amino acids were revealed. By substituting the revealed residues in XXT5 to those present at the same positions in XXT1, we anticipate to disrupt some protein‐protein interactions, thus reducing aggregation and increasing protein solubility. Vectors harboring the mutated gene were transformed into BL21 E. coli expression cells and, after expression and purification, mutant protein yield will be quantified using SDS‐PAGE. Those mutants that had higher expression in comparison with wild type XXT5 are currently being assessed using SEC analysis. The mutants showing higher solubility will be assayed for enzymatic activity to confirm the mutation did not affect protein activity. If the point mutation has shown sufficient expression, suitable solubility, and high activity, the screen for protein crystallization will be initiated.Solving the crystal structure of XXT1 and characterizing mutants allowed us to propose the model of xylosylation patterns in native xyloglucans. Structural information about the xyloglucan‐synthesizing enzymes is critical for our understanding of polysaccharide biosynthesis in plants and will have high impact on the development of plant biomass with improved properties for agriculture and industrial applications.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.