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.
|Number of pages||12|
|Journal||Protein Engineering, Design and Selection|
|Publication status||Published - 3 May 2018|
Bibliographical note© The Author(s) 2018. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details
This work was supported by the European Union and the EFPIA companies’ in kind contribution for the Innovative Medicine Initiative under Grant Agreement No. 115360 (Chemical manufacturing methods for the 21st century pharmaceuticals industries, CHEM21).
© The Author(s) 2018. Published by Oxford University Press.
- 3D-crystal structure
- active site mutation
- imine reductases
- β -hydroxyacid dehydrogenases