Sugar aminotransferases (SATs) catalyze the installation of chiral amines onto specific keto sugars, producing bioactive amino sugars. Their activity has been utilized in artificial reactions, such as using the SAT WecE to transform valienone into the valuable α-glucosidase inhibitor valienamine. However, the low thermostability and limited activity on non-natural substrates have hindered their applications. Simultaneously improving stability and enzyme activity is particularly challenging owing to the acknowledged inherent trade-off between stability and activity. A customized combinatorial active-site saturation test–iterative saturation mutagenesis (CAST-ISM) strategy was used to simultaneously enhance the stability and activity of WecE toward valienone. Fourteen hotspots related to improving the stability–\activity trade-off were identified based on evolutionary conservation and the average mutation folding energy assessment of 57 residues in the active site of WecE. Positive mutagenesis and combinatorial mutations of these specific residues were accomplished via site-directed saturation mutagenesis (SSM) and iterative evolution cycles. Compared with those of the wild-type (WT) WecE, the quadruple mutant M4 (Y321F/K209F/V318R/F319V) displayed a 641.49-fold increase in half-life (t1/2) at 40 °C and a 31.37-fold increase in activity toward the non-natural substrate valienone. The triple mutant M3 (Y321F/K209F/V318R) demonstrated an 83.04-fold increase in (t1/2) at 40 °C and a 37.77-fold increase in activity toward valienone. The underlying mechanism was dependent on the strengthened interface interactions and shortened transamination reaction catalytic distance, compared with those of the WT, which improved the stability and activity of the obtained mutants. Thus, we accomplished a general target-oriented strategy for obtaining stable and highly active SATs for artificial amino-sugar biosynthesis applications.

中文翻译：

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开发一种工程化糖转氨酶，同时提高稳定性和非天然底物活性，以合成葡萄糖苷酶抑制剂 Valienamine


糖氨基转移酶 （SAT） 催化手性胺安装在特定的酮糖上，产生生物活性氨基糖。它们的活性已被用于人工反应，例如使用 SAT WecE 将 valienone 转化为有价值的 α-葡萄糖苷酶抑制剂 valienamine。然而，低热稳定性和对非天然底物的有限活性阻碍了它们的应用。由于稳定性和活性之间公认的固有权衡，同时提高稳定性和酶活性尤其具有挑战性。使用定制的组合活性位点饱和度测试 - 迭代饱和诱变 （CAST-ISM） 策略来同时增强 WecE 对 valienone 的稳定性和活性。基于进化守恒和 WecE 活性位点 57 个残基的平均突变折叠能量评估，确定了 14 个与提高稳定性-活性权衡相关的热点。这些特定残基的阳性诱变和组合突变是通过定点定向饱和诱变 （SSM） 和迭代进化循环完成的。与野生型 （WT） WecE 相比，四重突变体 M4 （Y321F/K209F/V318R/F319V） 在 40 °C 时半衰期 （t1/2） 增加了 641.49 倍，对非天然底物 valienone 的活性增加了 31.37 倍。三重突变体 M3 （Y321F/K209F/V318R） 在 40 °C 时 （t1/2） 增加 83.04 倍，对 valienone 的活性增加 37.77 倍。与 WT 相比，其潜在机制依赖于增强的界面相互作用和缩短的转氨反应催化距离，从而提高了所获得突变体的稳定性和活性。 因此，我们完成了一种通用的目标导向策略，用于人工氨基糖生物合成应用获得稳定且高活性的 SAT。