Fig. 1: Ancestral sequence reconstruction of family 1 glycosidases (GH1) and assessment of ancestral stability.

a Bayesian phylogenetic tree of GH1 protein sequences using 150 representative sequences. Triangles correspond to four major well-supported clades (see supplemental Fig. S1 for nodal support) with common functions indicated. Numbers inside the triangles correspond to the number of sequences in each clade. Scale bar represents 0.5 amino acid replacements per site per unit evolutionary time. Reconstructed ancestral sequences were inferred at the labeled nodes and the protein at node 72 was exhaustively characterized. b Determination of the optimum temperature for the ancestral glycosidase (upper panel) using two different substrates 4-nitrophenyl-β-D-glucopyranoside (red) and 4-nitrophenyl-β-D-galactopyranoside (blue). v/[E]₀ stands for the rate over the total enzyme concentration. The lower panel shows a differential scanning calorimetry profile for the ancestral glycosidase. Clearly, the activity drop observed at high temperature (upper panel) corresponds to the denaturation of the protein, as seen in the lower panel. c Plot of enzyme optimum temperature versus living temperature of the host organism for modern family 1 glycosidases. Data (Supplementary Dataset 1) are derived from literature searches, as described in Methods. Horizontal and vertical bars are not error bars, but represent ranges of organismal living temperatures and enzyme optimum temperatures when provided in the literature. Color code denotes the organisms that published literature describes as hyperthermophiles, extreme thermophiles, thermophiles, mesophiles, psychrophiles; gray color is used for organisms that have not been thus classified (plants that live at moderate temperatures in most cases). The line is a linear-squares fit (T_OPT = 21.68 + 0.824T_LIVING). Correlation coefficient is 0.89 and p ∼ 8.8 × 10⁻⁴⁵ (probability that the correlation results from chance). An environmental temperature of about 52 °C can be estimated from the optimum temperature of the ancestral glycosidase.