New imine-reducing enzymes from ß-hydroxyacid dehydrogenases by single amino acid substitutions.

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, ß-hydroxyacid dehydrogenases (ßHADs) were identified as the dehydrogenases most similar to IREDs. To understand how active site differences in IREDs and ßHADs enable the reduction of predominantly C = N or C = O bonds respectively, we substituted amino acid residues in ßHADs with the corresponding residues from the R-IRED-Sr and were able to increase the promiscuous activity of ßHADs for C = N functions by a single amino acid substitution. Variants ßHADAt_K170D and ßHADAt_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 (ßHADAt_K170D/F_F231A) outcompeting already described IRED selectivity.