Crossing the Border: From Keto‐ to Imine Reduction in Short‐Chain Dehydrogenases/Reductases

Abstract

The family of NAD(P)H‐dependent short‐chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto‐reducing activity, yet negligible imine‐reducing capability, and mining the Short‐Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active‐site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine‐reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto‐reducing activity was engineered into a promiscuous enzyme with imine‐reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine‐reducing enzymes.

Systematic Evaluation of Imine‐Reducing Enzymes: Common Principles in Imine Reductases, β‐Hydroxy Acid Dehydrogenases, and Short‐Chain Dehydrogenases/ Reductases

“…As these principles might also apply to other NAD(P)H-dependent oxidoreductases, our model could support enzyme discovery, add to the diversity of imine-reducing enzymes, and contribute to rational enzyme engineering approaches toward imine reduction. [52]…”

Engineering of Thermostable β‐Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines

“…These flank the active site and mediate the local electrostatic fine‐tuning of the catalytic residues [36] . Exchanging several flanking residues in a dehydrogenase with distinct carbonyl‐reducing activity resulted in a promiscuous enzyme with imine‐reducing activity [37] . Recently, three new imine‐reducing enzymes were generated by introducing single‐point mutations into βHADs, including the exchange of the proton donor and the removal of a bulky gate‐keeping residue [38] .…”

“…[36] Exchanging several flanking residues in a dehydrogenase with distinct carbonyl-reducing activity resulted in a promiscuous enzyme with imine-reducing activity. [37] Recently, three new imine-reducing enzymes were generated by introducing singlepoint mutations into βHADs, including the exchange of the proton donor and the removal of a bulky gate-keeping residue. [38] To access imine reduction in the glyoxylate reductase from Arabidopsis thaliana (At-βHAD), the catalytically essential lysine at position 170 (K170) was exchanged by aspartic acid.…”

Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity

“…Furthermore, a SDR from Methylobacterium sp. 77 that only exhibits ketoreductase activity was recently engineered to gain imine reductase activity by introducing four mutations in its active site to resemble some of the structural features of the NR reductase [18] . Notably, in all of these cases, the ketoreductase and the imine reductase activities were strictly substrate dependent; furthermore, the SDR enzymes were active toward pre‐formed (cyclic) imines but reductive amination between a carbonyl compound and an amine donor was not reported.…”

“…77 that only exhibits ketoreductase activity was recently engineered to gain imine reductase activity by introducing four mutations in its active site to resemble some of the structural features of the NR reductase. [18] Notably,i na ll of these cases, the ketoreductase and the imine reductasea ctivities were strictly substrate dependent;f urthermore, the SDR enzymes were active toward preformed (cyclic) imines but reductive amination between ac arbonyl compound and an amine donor was not reported. Conversely, other groupsh ave independently reported that few imine reductases (IReds)a nd reductivea minases (RedAms) possess promiscuousk etoreductasea ctivity on very specific substrates such as tri-, di-andm ono-fluorinated acetophenones at the terminalcarbon position.…”