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 ( Hc GalB), determined at 2.97 Å resolution. Hc GalB 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 Hc GalB, 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. • Cryo-EM reveals a novel top-to-top hexameric architecture of HcGalB. • The confined catalytic cavity enhances transgalactosylation. • Hc GalB synthesizes C6 and C5 based GOS with novel prebiotic potential.
Sanità et al. (Wed,) studied this question.