The C-terminal lectin-like domain modulates the substrate specificity and transglycosylation activity of rice β-galactosidase1 ( <i>Os</i> BGal1)

Background β-galactosidases (EC 3.2.1.23) are glycosidases that release nonreducing terminal β- d -galactosyl residues from saccharides or glycosides. In plants, most characterized β-galactosidases belong to glycoside hydrolase family 35 (GH35) and function in cell wall remodeling. Over half of plant GH35 enzymes contain a C-terminal lectin-like domain absent in GH35 enzymes from other kingdoms, but its function remains unclear. In this work, we investigate the role of the C-terminal lectin-like domain by the effect of its deletion in rice β-galactosidase 1 ( Os BGal1) on its enzymatic function. Methods We expressed Os BGal1 and a truncated variant lacking the C-terminal domain ( Os BGal1ΔCter) in Pichia pastoris . Both enzymes were purified to homogeneity, with Os BGal1ΔCter exhibiting a higher level of N-glycosylation than the full-length enzyme. Kinetic analysis of p NP-glycosides evaluated substrate preferences. Molecular dynamics (MD) simulations of the predicted structures were used to visualize the effect of the C-terminal lectin-like domain. Results Os BGal1 showed high activity toward p NP-β- d -galactopyranoside and negligible activity toward alternative substrates, except for p NP-β- d -fucopyranoside. In contrast, Os BGal1ΔCter exhibited a 20-fold reduction in catalytic efficiency toward p NP-β- d -galactopyranoside but acquired significant activity toward p NP-β- d -glucopyranoside and higher activity for p NP-β- d -fucopyranoside. Kinetic analysis confirmed this broadened substrate profile, with the truncated enzyme showing higher turnover on glucoside, fucoside, and xyloside substrates. Functional assays demonstrated that Os BGal1 catalyzes transglycosylation, whereas Os BGal1ΔCter lost this activity. The predicted structures of Os BGal1 showed that the C-terminal domain is more than 30 Å from the active site, and MD simulations validated these structures by demonstrating their stability. This study reveals that removal of the C-terminal lectin-like domain of Os BGal1 alters substrate specificity and reduces transglycosylation activity, which may provide a basis for engineering GH35 enzymes with customized catalytic properties.