Abstract
Main conclusion
TBG4 recognize multiple linkage types substrates due to having a spatially wide subsite + 1. This feature allows the degradation of AGI, AGII, and AGP leading to the fruit ripening.
Abstract
β-galactosidase (EC 3. 2. 1. 23) catalyzes the hydrolysis of β-galactan and release of d-galactose. Tomato has at least 17 β-galactosidases (TBGs), of which, TBG 4 is responsible for fruit ripening. TBG4 hydrolyzes not only β-1,4-bound galactans, but also β-1,3- and β-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).
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Abbreviations
- TBG4:
-
Tomato β-galactosidase 4
- AGI:
-
Arabinogalactan I
- AGII:
-
Arabinogalactan II
- AGP:
-
Arabinogalactan protein
- GH:
-
Glycoside hydrolase
- WT_Gal:
-
Wild type_d-galactose complex structure
- E181A_β-1,3-Gal2 :
-
E181A_β-1,3-galactobiose complex
- E181A_β-1,4-Gal2 :
-
E181A_β-1,4-galactobiose complex
- E181A_β-1,6-Gal2 :
-
E181A_β-1,6-galactobiose complex
- CBM:
-
Carbohydrate-binding module
- DP:
-
Degree of polymerization
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Acknowledgements
The synchrotron radiation experiments were performed at SPring-8 under proposal number 2016B2500 and 2016B2728. We thank Dr. Toshiji Tada of the Graduate School of Science, Osaka Prefecture University, and Ms. Setsu Nakae, Faculty of Biosciences, Nagahama Institute of Bio-Science and Technology. We thank Dr. Jerry Ståhlberg and Mikael Gudmundsson of the Department of Molecular Sciences, Swedish University of Agricultural Sciences, for refinement of the structures and for discussing the X-ray crystal structures. We thank the beamline staff at BL38B1 of SPring-8 (https://www.spring8.or.jp) for providing data collection facilities and support. This work was partially supported by Japan Society for the Promotion of Science (JSPS) a Grant-in-Aid for Scientific Research on Innovative Areas to KI (No. 18H05494).
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Matsuyama, K., Kondo, T., Igarashi, K. et al. Substrate-recognition mechanism of tomato β-galactosidase 4 using X-ray crystallography and docking simulation. Planta 252, 72 (2020). https://doi.org/10.1007/s00425-020-03481-4
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DOI: https://doi.org/10.1007/s00425-020-03481-4