(820.1) Selection of GDP-fucose by active site residues in Arabidopsis thaliana SPY
Tuesday, April 5, 2022
12:30 PM – 1:45 PM
Location: Exhibit/Poster Hall A-B - Pennsylvania Convention Center
Poster Board Number: A434
Jenelle Rolli (St. Olaf College), Kate Desrochers (St. Olaf College), Jack Taylor (St. Olaf College), Erin NewRingeisen (St. Olaf College), Hadia Mohammadzadh (St. Olaf College), Cassandra Joiner (St. Olaf College)
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 or 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. To determine which of SPYs active site amino acids govern GDP-fucose selection, residues equivalent to those necessary for OGTs function were mutated and expressed in E. coli. Using the in vitro malachite green glycosyltransferase assay, the mutants’ activity was analyzed and compared to wildtype. One mutant displayed wild-type levels of glycosylation, while another exhibited partial activity. The remaining three mutants lost glycosylation activity, suggesting essentiality for GDP-fucose selection.
