CryoEM structural analysis of a thermophilic galactooligosaccharides-producer β-galactosidase unravels an uncommon oligomeric structure

: Thermostable β-galactosidases represent promising biocatalysts for lactose hydrolysis and production of structurally defined galacto-oligosaccharides (GOS). Here we report the cryo-EM structure of the glycoside hydrolase family 42 (GH42) β-galactosidase from Heyndrickxia coagulans MA-13 (HcGalB), determined at 2.97 Å resolution. HcGalB adopts a canonical tripartite architecture and assembles into a barrel-like homo-hexamer composed of two staggered trimers that interact in an unusual top-to-top configuration. This quaternary arrangement contributes not only to structural stability but also to the modulation of substrate channeling and catalytic properties. Molecular docking revealed a surface groove shaped by conserved aromatic residues that might guide the substrate towards the catalytic pocket. Moreover, the structural data provide a mechanistic rationale for the efficient transgalactosylation activity of HcGalB, which predominantly generates β (1 → 3)-linked GOS, along with β(1 → 6) and β(1 → 4) linkages, as confirmed by 2D Nuclear Magnetic Resonance. Overall, these findings expand the structural landscape of GH42 enzymes and identify architecture-specific determinants that can be leveraged to optimize GH42 catalysts for industrial and functional food applications.