title: Structural and functional analysis of tomato β-galactosidase 4: insight into the substrate specificity of the fruit softening-related enzyme.
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.
authors.affiliation: Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Eda
authors.affiliation: Application Laboratories, Rigaku Corporation, Akishima, Tokyo, 196-8666, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Faculty of Biology-Oriented Science and Technology, Kinki University, Kinokawa, Wakayama, 649-6493, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Ishimaru
authors.affiliation: Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
authors.forename: Toshiji
authors.initials: T
authors.lastname: Tada
mesh_terms: D018360:Crystallography, X-Ray; D005638:Fruit / Q000201:enzymology; D018551:Solanum lycopersicum / Q000201:enzymology*; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000072417:Protein Domains; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
chemical_list: D010940:Plant Proteins; D001616:beta-Galactosidase
keywords: GH35 family; Tomato; crystal structures; fruit softening; β-galactosidase