Co-crystal Structure of Thermosynechococcus elongatus Sucrose Phosphate Synthase With UDP and Sucrose-6-Phosphate Provides Insight Into Its Mechanism of Action Involving an Oxocarbenium Ion and the Glycosidic Bond
Co-crystal Structure of Thermosynechococcus elongatus Sucrose Phosphate Synthase With UDP and Sucrose-6-Phosphate Provides Insight Into Its Mechanism of Action Involving an Oxocarbenium Ion and the Glycosidic Bond
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DOI：
10.3389/fmicb.2020.01050
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发表时间：
2020-05-26
影响因子：
5.2
通讯作者：
Su, Jiyong
中科院分区：
文献类型：
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作者：
Li, Yuying;Yao, Yuan;Su, Jiyong
在绿色植物中，蔗糖可以帮助对抗非生物应激。蔗糖磷酸合成酶（SPS）是一种众所周知的蔗糖合成限速酶。然而，迄今为止，没有已知的晶体结构SPS从植物或蓝藻。在这项研究中，我们报道了长胞热聚球菌SPS与UDP和蔗糖-6-磷酸（S6P）的第一个共晶结构。在催化位点内，His158和Glu331的侧链以及来自UDP的两个磷酸基团与S6P中葡萄糖部分的四个羟基形成氢键。这种结合导致这四个羟基部分带负电荷，从而促进C1氧羰基离子的形成。His158和其中一个羟基之间的氢键断裂可能引发C1氧羰基离子和果糖-6-磷酸的C2羟基之间形成共价键。与我们的结构模型一致，我们观察到两个SPS突变体H158A和E331A失去了所有的催化活性。此外，SPS - a域中两个环（loop1和loop2）残基的电子密度未被观察到，这表明它们具有动态性质。b因子分析和全长酶和a结构域的分子动力学刺激表明，这两个环对底物和产物的结合和释放至关重要。此外，温度梯度分析表明，SPS在70℃时活性最高，表明该酶具有用于S6P工业生产的潜力。
In green species, sucrose can help antagonize abiotic stress. Sucrose phosphate synthase (SPS) is a well-known rate-limiting enzyme in the synthesis of sucrose. To date, however, there is no known crystal structure of SPS from plant or cyanobacteria. In this study, we report the first co-crystal structure of SPS from Thermosynechococcus elongatus with UDP and sucrose-6-phosphate (S6P). Within the catalytic site, the side chains of His158 and Glu331, along with two phosphate groups from UDP, form hydrogen bonds with the four hydroxyl groups of the glucose moiety in S6P. This association causes these four hydroxyl groups to become partially negatively charged, thus promoting formation of the C1 oxocarbenium ion. Breakage of the hydrogen bond between His158 and one of the hydroxyl groups may trigger covalent bond formation between the C1 oxocarbenium ion and the C2 hydroxyl of fructose-6-phosphate. Consistent with our structural model, we observed that two SPS mutants, H158A and E331A, lost all catalytic activity. Moreover, electron density of residues from two loops (loop1 and loop2) in the SPS A-domain was not observed, suggest their dynamic nature. B-factor analysis and molecular dynamics stimulations of the full-length enzyme and A-domain indicate that both loops are crucial for binding and release of substrate and product. In addition, temperature gradient analysis shows that SPS exhibits its highest activity at 70 degrees C, suggesting that this enzyme has the potential of being used in industrial production of S6P.