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

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Abstract

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.

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