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).

中文翻译：

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基于 X 射线晶体学和对接模拟的番茄 β-半乳糖苷酶 4 的底物识别机制

主要结论 TBG4 识别多种连锁类型的底物，因为它具有空间宽的子位点 + 1。该特征允许 AGI、AGII 和 AGP 降解导致果实成熟。摘要 β-半乳糖苷酶 (EC 3. 2. 1. 23) 催化 β-半乳聚糖的水解和 d-半乳糖的释放。番茄至少含有 17 种 β-半乳糖苷酶 (TBG)，其中 TBG 4 负责果实成熟。TBG4 不仅水解 β-1,4-结合的半乳聚糖，还水解 β-1,3- 和 β-1,6-半乳聚糖。在这项研究中，我们使用 X 射线晶体学、整体细化和对接模拟比较了每种酶-底物复合物，以了解 TBG4 的广泛底物特异性。在 subsite - 1 中，大多数相互作用在半乳糖二糖的每种连锁类型中都是保守的；但是，在 subsite + 1 中发现了一些差异，由于巨大的催化袋体积。除此之外，对接模拟表明 TBG4 可能有更多的阳性亚位点来识别和水解更长的半乳聚糖。总之，我们的研究结果表明，在番茄果实成熟过程中，TBG4 通过降解阿拉伯半乳聚糖 I (AGI)、阿拉伯半乳聚糖 II (AGII) 和阿拉伯半乳聚糖蛋白 (AGP) 的碳水化合物部分发挥重要作用。