We report the exploration of the evolutionary relationship between imine reductases (IREDs) and other dehydrogenases. This approach is informed by the sequence similarity between these enzyme families and the recently described promiscuous activity of IREDs for the highly reactive carbonyl compound 2,2,2-trifluoroacetophenone. Using the structure of the R-selective IRED from Streptosporangium roseum (R-IRED-Sr) as a model, β-hydroxyacid dehydrogenases (βHADs) were identified as the dehydrogenases most similar to IREDs. To understand how active site differences in IREDs and βHADs enable the reduction of predominantly C = N or C = O bonds respectively, we substituted amino acid residues in βHADs with the corresponding residues from the R-IRED-Sr and were able to increase the promiscuous activity of βHADs for C = N functions by a single amino acid substitution. Variants βHADAt_K170D and βHADAt_K170F lost mainly their keto acid reduction activity and gained the ability to catalyze the reduction of imines. Moreover, the product enantiomeric purity for a bulky imine substrate could be increased from 23% ee (R-IRED-Sr) to 97% ee (βHADAt_K170D/F_F231A) outcompeting already described IRED selectivity.

中文翻译：

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通过单个氨基酸取代从β-羟酸脱氢酶产生新的亚胺还原酶。

我们报告探索亚胺还原酶（IREDs）和其他脱氢酶之间的进化关系。这些酶家族之间的序列相似性和最近描述的IRED对高反应性羰基化合物2,2,2-三氟苯乙酮的混杂活性为这种方法提供了信息。以玫瑰链霉菌（R-IRED-Sr）的R选择性IRED的结构为模型，β-羟酸脱氢酶（βHADs）被鉴定为与IRED最相似的脱氢酶。要了解IRED和βHAD中活性位点的差异如何分别减少主要的C = N或C = O键，我们用来自R-IRED-Sr的相应残基取代了βHADs中的氨基酸残基，并且能够通过单个氨基酸取代来增加βHADs对C = N功能的混杂活性。变体βHADAt_K170D和βHADAt_K170F主要失去了酮酸还原活性，并具有催化亚胺还原的能力。此外，大体积亚胺底物的产物对映体纯度可从23％ee（R-IRED-Sr）增加到97％ee（βHADAt_K170D/ F_F231A），胜过已经描述的IRED选择性。