title: A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination.
abstract: Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration system. For applications of IREDs as cell-free catalysts, (i) we created an IRED variant showing an improved activity for NADH. With rational design we were able to identify four residues in the (R)-selective IRED from Streptomyces GF3587 (IR-Sgf3587), which coordinate the 2'-phosphate moiety of the NADPH cofactor. From a set of 15 variants, the highest NADH activity was caused by the single amino acid exchange K40A resulting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability using an immobilisate obtained either from purified enzyme or from lysate using the EziG(™) carriers. Applying the variant and NADH, we reached 88% conversion in a preparative scale biotransformation when employing 4% (w/v) 2-methylpyrroline. (iii) We demonstrated a one-enzyme cofactor regeneration approach using the achiral amine N-methyl-3-aminopentanone as a hydrogen donor co-substrate.
authors.affiliation: Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Gand
authors.affiliation: Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Thöle
authors.affiliation: Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany.
authors.forename: Hubertus
authors.initials: H
authors.lastname: Müller
authors.affiliation: Enzymicals AG, Walther-Rathenau-Straße 49a, 17489 Greifswald, Germany. Electronic address: henrike.brundiek@enzymicals.com.
authors.forename: Henrike
authors.initials: H
authors.lastname: Brundiek
authors.affiliation: Structural Biology Laboratory and Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand. Electronic address: g.bashiri@auckland.ac.nz.
authors.forename: Ghader
authors.initials: G
authors.lastname: Bashiri
authors.affiliation: Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany. Electronic address: Matthias.Hoehne@uni-greifswald.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Höhne
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003641:Deamination; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007097:Imines / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D007097:Imines; D011994:Recombinant Proteins; D009243:NAD; D010088:Oxidoreductases
keywords: Cofactor specificity; Coupled oxidative deamination; Imine reductase; Immobilization; Preparative scale biocatalysis; Rational design