The plant cell wall is primarily a polysaccharide mesh of the most abundant biopolymers on earth. Although one of the richest sources of biorenewable materials, the biosynthesis of the plant polysaccharides is poorly understood. Structures of many essential plant glycosyltransferases are unknown and suitable substrates are often unavailable for in vitro analysis. The dearth of such information impedes the development of plants better suited for industrial applications. Presented here are structures of xyloglucan xylosyltransferase 1 (XXT1) without ligands and in complexes with UDP and cellohexaose. XXT1 initiates side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. Structures here and the properties of mutant XXT1s are consistent with a SN-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. The XXT1 dimer alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed by the acceptor binding cleft. Homology modeling of XXT2 and XXT5, the other two xylosyltransferases involved in xyloglucan biosynthesis, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. An assembly of the three XXTs can produce the xylosylation patterns of native xyloglucans, suggesting the involvement of an organized multienzyme complex in the xyloglucan biosynthesis.
Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins, and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate Xyloglucan Xylosyltransferase 1 (XXT1), a GT involved in synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from UDP-Xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-Xylose and UDP-Glucose, while reactions with UDP-Arabinose and UDP-Galactose are over 10-fold slower. Using kcat/Km as a measure of efficiency, UDP-Xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-Glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific, and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems.
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.
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