Fig. 3: Structural features of the active site.

a Stereo view of the active site for the AM0627^E326A–P1–Zn²⁺ complex. The residues forming the active site are depicted as cyan (catalytic domain) and orange (IgG-fold domain) carbon atoms. The rest of the colors for the metal, the water molecule, and the dotted lines for the hydrogen bond interactions are the same as described in Fig. 2 . The GalNAc moieties are displayed in yellow according to the SNFG⁶⁰, while the Gal moieties are displayed in magenta for illustration purposes. The inset shows a scheme of the subsite nomenclature for the amino acids and the sugar moieties of P1. Note that the sugar moieties in the inset are displayed in yellow and with different symbols according to the SNFG. b Alternative close-up view of the active site to highlight the interactions between Tyr470 and the GalNAc and Gal moieties located at G1 and G2, respectively. c Time course of a cleavage assay using the 0.4 μM of wt AM0627^A21-E506 and the different mutants with P1 (57 μM) incubated at 37 °C. The cleavage was monitored by detecting the peak of substrate and product by MALDI spectra and the % of the remaining substrate and product formation was estimated. The raw spectra of MALDI-TOF analysis are given in Supplementary Fig. 3. All experiments were done in duplicate (n = 2 independent experiments). Source data are provided as a Source Data file. Note that each time point represents the average of two independent determinations.