Substrate-recognition mechanism of tomato β-galactosidase 4 using X-ray crystallography and docking simulation

beta-galactosidase (EC 3. 2. 1. 23) catalyzes the hydrolysis of beta-galactan and release ofd-galactose. Tomato has at least 17 beta-galactosidases (TBGs), of which, TBG 4 is responsible for fruit ripening. TBG4 hydrolyzes not only beta-1,4-bound galactans, but also beta-1,3- and beta-1,6-galactans. In this study, we compared each enzyme-substrate complex using X-ray crystallography, ensemble refinement, and docking simulation to understand the broad substrate-specificity of TBG4. In subsite - 1, most interactions were conserved across each linkage type of galactobioses; however, some differences were seen in subsite + 1, owing to the huge volume of catalytic pocket. In addition to this, docking simulation indicated TBG4 to possibly have more positive subsites to recognize and hydrolyze longer galactans. Taken together, our results indicated that during tomato fruit ripening, TBG4 plays an important role by degrading arabinogalactan I (AGI), arabinogalactan II (AGII), and the carbohydrate moiety of arabinogalactan protein (AGP).