New Imine Reducing Enzymes from β-Hydroxyacid Dehydrogenases by Single Amino Acid Substitutions

TY - JOUR

T1 - New Imine Reducing Enzymes from β-Hydroxyacid Dehydrogenases by Single Amino Acid Substitutions

AU - Lenz, Maike

AU - Fademrecht, Silvia

AU - Sharma, Mahima

AU - Pleiss, Juergen

AU - Grogan, Gideon James

AU - Nestl, Bettina

N1 - © 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 Funding Information: 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). Publisher Copyright: © The Author(s) 2018. Published by Oxford University Press.

PY - 2018/5/3

Y1 - 2018/5/3

N2 - 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.

AB - 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.

KW - 3D-crystal structure

KW - active site mutation

KW - imine reductases

KW - promiscuity

KW - β -hydroxyacid dehydrogenases

UR - http://www.scopus.com/inward/record.url?scp=85052642476&partnerID=8YFLogxK

U2 - 10.1093/protein/gzy006

DO - 10.1093/protein/gzy006

M3 - Article

SN - 1741-0126

VL - 31

SP - 109

EP - 120

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

IS - 4

ER -