The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase β-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multistep routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogues of the amino acid tryptophan (Trp), which are frequently used in pharmaceutical synthesis as well as chemical biology. However, certain valuable compounds, such as the blue fluorescent amino acid 4-cyanotryptophan (4-CN-Trp), could only be made in low yield, even at elevated temperature (75 °C). Here, we describe the engineering of TrpB from Thermotoga maritima that improved synthesis of 4-CN-Trp from 24% to 78% yield. Remarkably, although the final enzyme maintains high thermostability (T₅₀ = 93 °C), its temperature profile is shifted such that high reactivity is observed at ∼37 °C (76% yield), creating the possibility for in vivo 4-CN-Trp production. The improvements are not specific to 4-CN-Trp; a boost in activity at lower temperature is also demonstrated for other Trp analogues.

中文翻译：

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改进的合成4-氰基色氨酸和其他色氨酸类似物在水溶液中使用来自栖热菌的TrpB变体

随着化学家努力减少对有机溶剂的依赖，转而使用对环境更有益的水性介质，酶在合成中的使用已越来越广泛。考虑到这一点，我们以前致力于工程改造色氨酸合酶β-亚基（TrpB），以生产以前通过涉及水敏试剂的多步路线合成的非规范氨基酸。事实证明，该酶促平台可有效合成氨基酸色氨酸（Trp）的类似物，而该氨基酸通常用于药物合成和化学生物学领域。但是，某些有价值的化合物，例如蓝色荧光氨基酸4-氰基色氨酸（4-CN-Trp），即使在升高的温度（75°C）下也只能以低产率制备。在这里，我们描述了TrpB的工程maritoma可将4-CN-Trp的合成从24％提高到78％。值得注意的是，尽管最终的酶保持了较高的热稳定性（T ₅₀ = 93°C），但其温度曲线发生了变化，以致于在〜37°C时观察到高反应性（产率为76％），从而为体内4-CN-色氨酸生产。改进不是特定于4-CN-Trp的。对于其他Trp类似物，在较低温度下的活性也得到了提高。