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oai:figshare.com:article/33971371 2026-09-23T06:18:07Z category_8 portal_316 item_type_3 month_year_09_2026

Supplementary file 1_A C39-like protein from methanogenic archaea: structural insights and functional divergence in β-galactose analogue degradation.docx Leizhou Guo (25094794) Liuyan Wang (10693388) Liping Bai (1666480) Microbiology C39-like protein glycosidase activity methanogenic archaea structure and function β-Galactose analogs <p>Methanogenic archaea harbor diverse enzymes that support their survival in extreme environments and play key roles in global carbon cycling. 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. Molecular docking and site-directed mutagenesis further implicate Loop 3 (residues D314–D324) as a critical determinant of substrate recognition, with the C316-C322 disulfide bond likely stabilizing its conformation. Collectively, these findings expand the functional repertoire of the C39 peptidase superfamily, suggest an evolutionary connection between peptidases and glycosidases, and establish A0A126R2S0 as a candidate biocatalyst for β-galactoside processing.</p> 2026-09-23T06:18:07Z Dataset Dataset 10.3389/fmicb.2026.1895254.s001 https://figshare.com/articles/dataset/Supplementary_file_1_A_C39-like_protein_from_methanogenic_archaea_structural_insights_and_functional_divergence_in_-galactose_analogue_degradation_docx/33971371 CC BY 4.0