Structure-Based Design of Small Imine Reductase Panels for Target Substrates

Biocatalysis is important in the discovery, development, and manufacture of pharmaceuticals. However, the identification of enzymes for target transformations of interest requires major screening efforts. Here, we report a structure-based computational workflow to prioritize protein sequences by a score based on predicted activities on substrates, thereby reducing a resource-intensive laboratory-based biocatalyst screening. We selected imine reductases (IREDs) as a class of biocatalysts to illustrate the application of the computational workflow termed IREDFisher. Validation by using published data showed that IREDFisher can retrieve the best enzymes and increase the hit rate by identifying the top 20 ranked sequences. The power of IREDFisher is confirmed by computationally screening 1400 sequences for chosen reductive amination reactions with different levels of complexity. Highly active IREDs were identified by only testing 20 samples in vitro. Our speed test shows that it only takes 90 min to rank 85 sequences from user input and 30 min for the established IREDFisher database containing 591 IRED sequences. IREDFisher is available as a user-friendly web interface (https://enzymeevolver.com/IREDFisher). IREDFisher enables the rapid discovery of IREDs for applications in synthesis and directed evolution studies, with minimal time and resource expenditure. Future use of the workflow with other enzyme families could be implemented following the modification of the workflow scoring function.


Table of Contents
Table S1.Hit rate in 20 sequences by IREDFisher and random selection.As a control, 20 sequences were randomly selected from the panels 1000 times and the average hit rate was calculated.The margin of error was calculated based on 95% confidence levels.Table S3.Retention times from GC-MS analysis for test reaction 18.

Compound Retention time (minutes)
Cycloheptanone ( 31 Table S6.Retention times from HPLC analysis for test reaction 25.

Figure S1 .Figure S2 .Figure S3 .Figure S7 .Figure S9 .Figure S11 . 28 Figure S12 .
Figure S1.Examples of pharmaceutically relevant 4-formylbenzoic acid benzaldehyde derivatives Figure S2.Comparison of imine reductase structures predicted by homology modelling and AlphaFold.Analytical methods Figure S3.Scheme of reductive amination of cyclohexanone with cyclopropylamine for the synthesis of 18 Figure S4.GC-MS spectra from screening for the synthesis of 18 Table S3.Retention times from GC-MS analysis for test reaction 18 Figure S5.Scheme of reductive amination of chlorocyclohexanone with cyclopropylamine for the synthesis of 26 Figure S6.GC-MS spectra from screening for the synthesis of reaction 26 Table S4.Retention times from GC-MS analysis for test reaction 26 Figure S7.Scheme of reductive amination of cycloheptanone with propargylamine for the synthesis of 27 Figure S8.GC-MS spectra from screening for the synthesis of 27 13 Table S5.Retention times from GC-MS analysis for test reaction 27 Figure S9.Scheme of reductive amination of benzaldehyde with cyclopropylamine for the synthesis of 25 Figure S10.LC/MS chromatograms from screening for the synthesis of 25Table S6.Retention times from HPLC analysis for test reaction 25 Figure S11.Scheme of reductive amination of 4-formylbenzoic acid with cyclopropylamine for synthesis of 28 Figure S12.HPLC chromatograms from screening for the synthesis of compound 28 Table S7.Retention times from HPLC analysis for test reaction 28 IRED Sequences

Figure S2 .
Figure S2.Structural comparison of an imine reductase predicted by homology modelling (in magenta) and AlphaFold (in green).Left panel, the overall structure.Right panel, a close-up view of amino acids in the substrate-binding site.Side chains in the active site were aligned, resulting in a root mean square deviation about 1.02 Å.

Figure S3 .Figure S4 .
Figure S3.Scheme of reductive amination of cyclohexanone with cyclopropylamine for the synthesis of 18. Cofactor regeneration system consists of GDH, NADP + and glucose.Test reaction 18 chromatograms a)

Figure S7 .Figure S8 .
Figure S7.Scheme of reductive amination of cycloheptanone with propargylamine for the synthesis of 27.Cofactor regeneration system consists of GDH, NADP + and glucose.Test reaction 27 chromatograms

Figure S9 .Figure S10 .
Figure S9.Scheme of reductive amination of benzaldehyde with cyclopropylamine for the synthesis of 25.Cofactor regeneration system consists of GDH, NADP + and glucose.Test reaction 25 chromatograms a)

Table S1 .
Hit rate in 20 sequences by IREDFisher and random selection

Table S2 .
Likelihood to retrieve the best hit by IREDFisher and Random selection

Table S2 . Likelihood to retrieve the best hit by IREDFisher and Random selection.
In most cases, IREDFisher was able to retrieve the best hit(s) from the whole panel.In some cases where the best hit did not appear, the second best hit was found.By comparison, random selection has a much lower chance of obtaining the best hit(s).

Table S7 .
Retention times from HPLC analysis for test reaction 28.