A methanogen hydrolase reveals the structure of archaeal peptidoglycan

Peptidoglycan (PG) is a near-universal and essential feature of bacterial cell walls and a major antimicrobial target¹. Although archaea generally lack PG², a PG-like polymer (pseudomurein or archaeal PG) was described decades ago in a major clade of methanogenic archaea^3-8, yet it has remained poorly characterized, owing to the lack of dedicated analytical tools. Here we identify and characterize ArmA from Methanobrevibacter smithii, a dominant member of the human gut microbiome⁹, as the first glycosyl hydrolase specific for archaeal PG. ArmA-mediated digestion reveals an unexpected architecture that revises the prevailing model of archaeal PG. The glycan backbone comprises N-acetylglucosamine/N-acetylgalactosamine linked to a previously undescribed sugar, which we name N-acetylarmosamine. Glycan strands alternate β(1,4) and β(1,3) linkages, and the stem peptide is attached by means of an amide bond to the N-acetylarmosamine succinyl group. ArmA has dual enzymatic activity, cleaving both glycosidic linkages and peptide crosslinks. Phylogenetic analyses show that ArmA homologues are restricted to PG-bearing archaea, and we confirm activity across diverse methanogens. We further demonstrate that ArmA is required to complete cytokinesis, cleaving archaeal PG at the site of cell division. Together, these findings overturn a 50-year-old paradigm on archaeal PG structure and establish ArmA as a critical tool that parallels the impact of muramidases in bacteria, enabling biochemical and genetic interrogation of methanogen cell-wall biology. Finally, given the ecological and biotechnological importance of methanogens^10,11, our results open new avenues for targeted intervention.