Supplementary file 1_A C39-like protein from methanogenic archaea: structural insights and functional divergence in β-galactose analogue degradation.docx

The C39 peptidase family, known for cleaving peptide substrates, now gains functional expansion through A0A126R2S0, a novel enzyme that shares structural homology with C39 peptidases (RMSD: 2.0–2.5 Å) but exhibits glycosidase activity. The enzyme harbors a conserved Q-C-H motif in which the third residue of the canonical catalytic triad is substituted from aspartate to asparagine.

Enzymatic assays demonstrate that A0A126R2S0 specifically hydrolyzes β-D-galactose analogs, such as p-nitrophenyl-β-D-galactopyranoside (PNPG, Sub 5), with an activity of 16.15 ± 0.71 μM/h, which is 7.8-fold higher than that toward α-configured analogs. Notably, it shows no detectable activity toward double-glycine peptides or archaeal cell wall polypeptides, distinguishing it from canonical C39 peptidases. Truncation experiments confirm that the C-terminal domain alone is sufficient for this glycosidase activity.