Internal Water Dynamics Control the Transglycosylation/Hydrolysis Balance in the Agarase (AgaD) of Zobellia galactanivorans
Internal Water Dynamics Control the Transglycosylation/Hydrolysis Balance in the Agarase (AgaD) of Zobellia galactanivorans
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DOI：
10.1021/acscatal.7b00348
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发表时间：
2017-05-01
期刊：
影响因子：
12.9
通讯作者：
Tellier, Charles
中科院分区：
文献类型：
--
作者：
David, Benoit;Irague, Romain;Tellier, Charles
在保留糖苷水解酶（GH）中，由于在水中有利的天然水解活性，转糖基酶活性通常较低。提高这些酶的相对转糖基酶活性对于获得适合于合成寡糖的酶是特别有意义的。我们探讨了工程内的水动力学的内切β-琼脂糖酶AgaD的转糖基化/水解（T/H）平衡的影响。通过突变三个氨基酸（D341，Q342和S351），这可以控制水进入一个假定的水通道结束接近活性位点，我们获得了AgaD变体与倒置的T/H平衡。对于最佳突变体D341 L/Q342 H/S351 F，与野生型相比水解活性降低了50倍，而转糖基酶活性保持甚至略有提高。该变体通过与十琼脂糖作为底物的缩合反应产生大量的寡琼脂糖。分子动力学模拟表明，这些酶的修饰与更高的水动力学，揭示了水的存活时间显着减少，并减少在一个通道中的水的清洗时间结束接近活性位点。这些结果表明，修改GH中的水动力学可能是转糖基酶活性工程的合理基础。
In retaining glycoside hydrolases (GHs), transglycosylase activity is often low due to the natural hydrolytic activity that is favored in water. Improving the relative transglycosylase activity of these enzymes is of particular interest to obtain enzymes suitable for the synthesis of oligosaccharides. We explored the effect of engineering the water dynamics within the endo-beta-agarase AgaD on the transglycosylation/hydrolysis (T/H) balance. By mutating three amino acids (D341, Q342, and S351), which could control water access to a putative water channel ending close to the active site, we obtained AgaD variants with an inverted T/H balance. For the best mutant, D341L/Q342H/S351F, the hydrolysis activity was reduced 50-fold in comparison to the wild type, while the transglycosylase activity was maintained and even slightly improved. This variant produced a large amount of oligo-agaroses by a disproportionation reaction with decaagarose as the substrate. Molecular dynamics simulations showed that these enzymatic modifications were correlated with higher water dynamics, as revealed by a marked reduction in the water survival time and a decrease in the purge time of water in a channel ending close to the active site. These results suggest that modifying the water dynamics in GHs could be a rational basis for engineering of transglycosylase activity.