O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) transfers O-GlcNAc from the nucleotide-sugar UDP-GlcNAc to thousands of nuclear and cytoplasmic proteins. OGT is an essential enzyme, and misregulation of O-GlcNAcylation has been linked to cancers and<br/>neurodegeneration. Most eukaryotic organisms possess a single structural homolog of OGT. However, Arabidopsis thaliana has two: Spindly (SPY) and Secret Agent (SEC). Whilst being structurally similar, SPY and SEC select different nucleotide-sugars, GDP-fucose and UDP-GlcNAc, respectively. Using human OGT’s crystal structure, we made corresponding active site mutations in AtSPY that we hypothesize are critical for GDP-fucose binding and glycosyltransferase activity. Here, we recombinantly expressed each AtSPY mutant in E. coli and purified them using a two-step purification method. The catalytic activity of each mutant was tested using an in vitro malachite green glycosyltransferase assay to determine whether OGT’s catalysis mechanism is conserved in AtSPY and identify the active site residues that govern GDP-fucose selection.

Selection of GDP-fucose by active site residues in <i>Arabidopsis thaliana </i>SPY

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Selection of GDP-fucose by active site residues in Arabidopsis thaliana SPY