C-terminal domains of β-galactosidase from Paenibacillus macquariensis modulate product distribution by altering...

GH2 β-galactosidases synthesize galacto-oligosaccharides (GOS) with various degrees of polymerization and linkage types. For some GH2 multidomain β-galactosidases, catalytic efficiency and size of product oligosaccharides can be modified by truncating the C-terminal domains. Yet, the impact of C-terminal truncation on product linkage distribution remains unexplored, and the mechanisms behind this strategy are not entirely understood. Investigating how C-terminal truncation affects GOS synthesis is important for producing desired product structures. Herein, we expressed the GH2 β-galactosidase PmGal and analyzed the product distribution of both the wild-type enzyme and its C-terminally truncated forms. One of these variants showed enhanced specific activity and increased allolactose productivity. Through molecular dynamics analysis, we examined the functional roles of the C-terminal domains. Our findings reveal that truncation increases the flexibility of both the active-site loops in the catalytic domain and the surface loop in the C-terminal domain via dynamic allostery. The enhanced flexibility alters the relative positioning of the C-terminus and catalytic domain, and influences substrate binding conformation, resulting in a shift in product distribution. Overall, our study provides valuable insights into truncation strategies for controlling transgalactosylation efficiency and product distribution. It also enhances our understanding of the structural factors influencing β-galactosidase catalysis.