Recently, significant public and private investments are being made to support research into elucidating biosynthetic pathways towards the formation of plant cell wall glycans. Genetically engineered plants with modified cell wall composition represent a promising strategy to enhance the economic viability of exploiting biomass as a renewable resource. However, a prerequisite to do so is a detailed knowledge of the molecular structure of the cell wall and the biosynthetic enzymes responsible for its construction. Plant cell wall polysaccharides are synthesized by numerous enzymes located in the plasma membrane and the Golgi apparatus. The most important group of enzymes is the glycosyltransferases which catalyze the transfer of sugar nucleotide donors to suitable acceptor substrates. The genome of the reference plant <i>Arabidopsis thaliana</i> encodes more than 450 glycosyltransferases. Of these, about 100 glycosyltransferases are involved glycosylation of small molecules, whereas the large majority of the glycosyltransferases has been proposed to catalyze the biosynthesis of plant cell wall polysaccharides. However, only few of the corresponding enzymes have been biochemically characterized, due to a lack of suitable assays.<br/><br/>We present here a glycan array-based assay for determining the substrate specificities of plant glycosyltransferases. This array is equipped with a unique collection of more than 70 plant cell wall oligosaccharides that we have prepared by automated solid-phase synthesis (automated glycan assembly). Chemically synthesized azido-functionalized sugar nucleotides are applied to the array and any incorporated monosaccharide is then selectively visualized by reaction with an alkyne-functionalized fluorescent dye. Using this assay, we were able to confirm and correct the specificities of known plant glycosyltransferases as well as identify new glycosyltransferases.

Characterizing plant glycosyltransferases in high throughput using azido-functionalized sugar nucleotides on glycan arrays

2019 Spring National Meeting

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