Structure and Activity of NADPH‐Dependent Reductase Q1EQE0 from Streptomyces kanamyceticus, which Catalyses the R‐Selective Reduction of an Imine Substrate

Abstract

NADPH-dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2-methyl-1-pyrroline to the chiral amine (R)-2-methylpyrrolidine with >99% ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 Å. Q1EQE0 functions as a dimer in which the monomer consists of an N-terminal Rossman-fold motif attached to a helical C-terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C-terminal domains are swapped, with a substrate-binding cleft formed between the N-terminal subunit of monomer A and the C-terminal subunit of monomer B. The structure is related to those of known β-hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de)protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme.

Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment

“…Consistent with the potential role of RedE in the reduction of pyrole ring in 2 , the closest characterized relative of the RedE reductase is the NADPH-dependent oxidoreductase Q1EQE0 that catalyzes the reduction of 2-methyl-1-pyrroline to (R)-2-methylpyrrolidine. 38 Based on these observations, a biosynthetic scheme for 2 was developed (Figure 5). Homologs of RedO, RedD, and RedP are known to produce the indolocarbazole intermediate 4 , which spontaneously decarboxylates to yield 5, 6 , and 7 in the absence of any other biosynthetic enzymes (Figure 3C).…”

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

“…Except for the serine dehydrogenase (Tchigvintsev et al, 2012), all of these enzymes are NADPH-dependent. The present work focuses on IR-Sgf3587 as model enzyme; however, due to the lack of a crystal structure, we generated a homology model based on the crystal structure of IR-Ska (Rodríguez-Mata et al, 2013). This enzyme (PDB-code 3ZHB) shares 53% sequence identity (71% similarity) with IR-Sgf3587.…”

“…13.9.8). This model and the crystal structure of IRSka (PDB-code 3ZHB, (Rodríguez-Mata et al, 2013)) were used to determine the binding pocket for the cofactor.…”

“…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.…”