Fig. 4: Active site and catalytical mechanism of SPY.

a Cartoon rendering of the active site in the ‘catalytic SPY’/GDP/‘substrate SPY’ complex. GDP is shown as stick. The color coding is the same as in Fig. 2a. b Close-up view of the active site in the ‘catalytic SPY’/GDP/‘substrate SPY’ complex. Residues of interest are shown as sticks. For simplicity, the side chains of I722, L723, T747 and T749 are omitted as they stabilize the GDP with their backbones. Residues in the ‘catalytic SPY’ and the substrate loop are labeled in black and red, respectively. Black dotted lines indicate hydrogen bonds. c Self-fucosylation of wild type SPY and mutants carrying point mutations in the active site. Upper panel (long exposure) and middle panel (short exposure) show western blotting with biotinylated AAL. Lower panel shows Coomassie blue staining. Experiments were independently repeated three times with similar results. Uncropped images of Western blotting membranes and gels are available as source data. d Fucosylation of DELLA by wild type SPY and mutants carrying point mutations in the active site. Upper panel (long exposure) and middle panel (short exposure) show western blotting with biotinylated AAL. Lower panel shows Coomassie blue staining. Experiments were independently repeated three times with similar results. Uncropped images of Western blotting membranes and gels are available as source data. e Proposed catalytic mechanism of SPY. The peptide is depicted in yellow loop with the reactive serine shown as stick. The α-phosphate in the GDP-fucose could act as the catalytic base. H495 probably contributes to maintain the structure of the active site. S496 stabilizes the orientation of the reactive serine and the substrate loop. K665 and T748 stabilize the GDP moiety. Y744 potentially stabilize the fucose moiety.