Fig. 2: 3D Structure of the ancestral glycosidase as determined by X-ray crystallography.

a Comparison between the ancestral structure determined in the absence (left) and presence (middle) of bound heme (red) and a homology model constructed as described in Supporting Information. The visual comparison reveals the missing sections in the electronic density of the ancestral protein, mostly in the protein without heme bound. b 3D structure of the ancestral protein without and with heme bound color-labeled according to normalized B-factor value and profiles of normalized B-factor versus residue number for the ancestral protein without (red) and with (blue) bound heme. Values are not shown for the sections that are missing in the experimental structures. c Proteolysis experiments with the ancestral glycosidase and the modern glycosidase from Halothermothrix orenii. The major fragments are labeled a, b, c and d. Molecular weights (MW) are shown for the markers used. Five independent experiments were performed with similar results. Mass spectrometry of the fragments predicts cleavage points within the red labeled sections in the shown structure. d Superposition of the structure of the ancestral glycosidase with that of the modern glycosidase from Halothermothrix orenii showing the critical active-site residues. e Superposition of the structures of the ancestral glycosidase without and with heme bound showing the critical active-site residues. In both (d) and (e), the highlighted active-site residues include the catalytic carboxylic acid residues (blue) and the residues involved in binding of the glycone (yellow) and aglycone (green) parts of the substrate.