Direct Reductive Amination of Ketones: Structure and Activity of S‐Selective Imine Reductases from Streptomyces

Abstract

The importance and structural diversity of chiral amines is well‐demonstrated by the myriad nonenzymatic methods for their chemical production. In nature, the production of amines is performed by transamination rather than by reduction of an imine precursor derived from the corresponding ketone. Imine reductases, however, show great potential in the reduction of cyclic imines that are stable towards hydrolysis in aqueous reaction media. Here, we report the catalytic activity of two S‐selective imine reductases towards 3,4‐dihydroisoquinolines and 3,4‐dihydro‐β‐carbolines and their activity in the direct reductive amination of ketone substrates. The crystal structures of the enzyme from Streptomyces sp. GF3546 in complex with the cofactor NADPH and from Streptomyces aurantiacus in native form have been solved and refined to a resolution of 1.9 Å.

Structure, Activity and Stereoselectivity of NADPH‐Dependent Oxidoreductases Catalysing the S‐Selective Reduction of the Imine Substrate 2‐Methylpyrroline

“…The distance between, for example, the Cα atoms of Val138 and Met192, which are on opposite faces of the NADPH binding cleft on either side of the plane of the side chain Tyr188, closes from 12.6 to 11.8 Å. Such a perturbation was also observed for ( S )‐IRED by Müller and co‐workers17 and explains in part the larger rmsd for 3546‐IRED in relation to Bc IRED, against the value in relation to Q1EQE0, in which no such movement was observed. On binding of NADPH, the side chain of Ser113 disengages from H‐bonding contact with the side chain of Gln189 and His262 and rotates 180° to form an H‐bond with the O2D oxygen of the NADPH ribose (at a distance of 3.0 Å), and also a weak H‐bond (3.7 Å) with the phenolic hydroxy group of Tyr188.…”

“…The selection of multiple alternative enzymes for structural studies proved fruitful in respect of the acquisition of structures of IREDs that were S ‐selective for the reduction of 1 . A structure of 3546‐IRED to a resolution of 3 Å was obtained, but during the preparation of this manuscript a structure at a superior resolution of 1.9 Å was published by Müller and co‐workers17 (PDB ID: 4OQY), so our data are not included here. The structure of Bc IRED was determined in two forms: an apo form to a resolution of 1.74 Å and a complex with NADPH that was determined to a resolution of 1.81 Å.…”

“…Now that the structure of an IRED that is R ‐selective for the reduction of 2MPN has been solved, it would be of interest to compare this to structures of homologues that are S ‐selective for reduction of this substrate. The structure of 3546‐IRED has recently been reported,17 but neither a detailed analysis of the active site, nor mutational studies on this enzyme as a representative member of enzymes with S selectivity towards 2MPN were presented. In this report we describe further studies on IREDs that catalyse the S ‐selective reduction of 2MPN, including the structures and activities of enzymes from Bacillus cereus BAG3X2‐2 ( Bc IRED) and Nocardiopsis halophila ( Nh IRED).…”

A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination

“…These enzymes allow two "types" of reactions: (i) the reduction of a cyclic imine to a cyclic sec-amine or (ii) the reductive amination of a ketone with a primary amine yielding a sec-amine, where a proof of concept (Huber et al, 2014;Scheller et al, 2015) and preparative applications were demonstrated very recently (Wetzl et al, 2016). In the previous years, several research groups discovered a large number of IREDs (Gand et al, 2014;Huber et al, 2014;Li et al, 2015;Man et al, 2015;Mitsukura et al, 2013Mitsukura et al, , 2011Mitsukura et al, , 2010Rodríguez-Mata et al, 2013;Scheller et al, 2015;Wetzl et al, 2015). In addition, an imine reductase engineering database with more than 1000 putative IREDs is established (Scheller et al, 2014).…”

“…In addition, an imine reductase engineering database with more than 1000 putative IREDs is established (Scheller et al, 2014). Until now the crystal structures of six different IREDs have been solved (Gand et al, 2014;Huber et al, 2014;Man et al, 2015;Rodríguez-Mata et al, 2013). All hitherto discovered IREDs of this superfamily show a strong preference for NADPH as the cofactor.…”