l-Threonine aldolases (l-TAs) catalyze the aldol condensation of aldehyde and glycine, offering direct enzymatic synthesis of β-hydroxy-α-amino acids under mild conditions. However, this method suffers from moderate yield and low stereoselectivity at the β-carbon. Given the importance of 4-(methylsulfonyl)phenylserine for the synthesis of florfenicol and thiamphenicol, mutations of a l-threonine aldolase from Pseudomonas sp. (l-PsTA) were performed in this study by error-prone PCR and combinatorial mutation. Some beneficial mutants were obtained by screening the mutant library using a stepwise visual method. 4-(Methylsulfonyl)phenylserine was synthesized in up to 71 % diastereomeric excess (de), which are much higher than the previously reported 2 % de value, by using the newly identified mutants. The mutants V200I and C187S/V200I were found to improve the product yield and stereoselectivity for the aldol condensation of various benzaldehydes with glycine. These results show that the amino acid residues outside of the substrate-binding cavity of l-PsTA play an important role in determining its Cβ-stereoseletivity.

中文翻译：

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L-苏氨酸醛缩酶工程用于制备 4-（甲基磺酰基）苯基丝氨酸，这是合成氟苯尼考和噻吩尼考的重要中间体

l-苏氨酸醛缩酶 (l-TAs) 催化醛和甘氨酸的醛缩缩合，在温和条件下提供 β-羟基-α-氨基酸的直接酶促合成。然而，这种方法的收率适中，β-碳的立体选择性较低。鉴于 4-（甲基磺酰基）苯基丝氨酸对合成氟苯尼考和噻吩菌素的重要性，来自假单胞菌属的 l-苏氨酸醛缩酶的突变。(l-PsTA) 在本研究中通过易错 PCR 和组合突变进行。通过使用逐步视觉方法筛选突变体库，获得了一些有益的突变体。通过使用新鉴定的突变体，4-(甲基磺酰基)苯基丝氨酸以高达 71% 的非对映体过量 (de) 合成，这远高于之前报道的 2% de 值。发现突变体 V200I 和 C187S/V200I 提高了各种苯甲醛与甘氨酸的羟醛缩合的产物产率和立体选择性。这些结果表明 l-PsTA 底物结合腔外的氨基酸残基在决定其 Cβ 立体选择性方面起着重要作用。