Structural and functional analysis of tomato β-galactosidase 4: insight into the substrate specificity of the fruit softening-related enzyme

    loading  Checking for direct PDF access through Ovid


Plant β-galactosidases hydrolyze cell wall β-(1,4)-galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β-galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β-(1,4)-galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1–7 that belongs to the β-galactosidase/exo-β-(1,4)-galactanase subfamily. The enzyme can hydrolyze a wide range of plant-derived (1,4)- or 4-linked polysaccharides, and shows a strong ability to attack β-(1,4)-galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β-D-galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β-sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β-galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β-(1,6)-galactobiose, and ˜0.6-fold activity towards β-(1,4)-galactobiose, compared with wild-type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β-galactosidases towards β-1,4 and β-1,6 linkages.

Significance Statement

Tomato β-galactosidases (TBG1-TBG7) in the glycosyl hydrolase 35 (GH35) family are thought to play important roles during fruit development and maturation. Here we report the crystal structure of TBG4 in complex with its substrate.

Related Topics

    loading  Loading Related Articles