The rapidly growing area of asymmetric imine reduction by imine reductases (IREDs) has provided alternative routes to chiral amines. Here we report the expansion of the reaction scope of IREDs by showing the stereoselective reduction of 2,2,2-trifluoroacetophenone. Assisted by an in silico analysis of energy barriers, we evaluated asymmetric hydrogenations of carbonyls and imines while considering the influence of substrate reactivity on the chemoselectivity of this novel class of reductases. We report the asymmetric reduction of C=N as well as C=O bonds catalysed by members of the IRED enzyme family.

Cell-free transcription-translation systems offer an effective and versatile platform to explore the impact of genetic variations on protein function. We have developed a protocol for preparing linear, mutagenic DNA templates for direct use in the PURE system, enabling the fast and semi-quantitative evaluation of amino acid variations on catalytic activity and stereo-selectivity in native and engineered variants of Old Yellow Enzyme.

In protein engineering, cell-free transcription/translation of linear mutagenic DNA templates can tremendously accelerate and simplify the screening of enzyme variants. Using the RApid Parallel Protein EvaluatoR (RAPPER) protocol, we have evaluated the impact of amino acid substitutions and loop truncations on substrate specificity and stereoselectivity of Old Yellow Enzyme 1 from Saccharomyces pastorianus. Our study demonstrates the benefit of systematically assessing amino acid variations including substrate profiling to explore sequence-function space.

The cover picture shows a recently discovered imine reductase (IRED) planet in the outer space of enzyme biocatalysts. The carbonyl‐reducing enzyme family of β‐hydroxyacid dehydrogenases (βHAD) orbits the planet as blue moon demonstrating their evolutionary relatedness to IREDs. We investigated the promiscuity of IREDs for ketone reduction by elucidating the electronic effects of substituents on substrate molecules and their influence on catalysis. The IRED activity for the examined substrates is illustrated by the distance of the constellations to the planet. The authors identified a “substrate rocket” by substitution of the keto functionality by a trifluoromethyl group. We are grateful to Richard Mensah for the graphic design of the cover. More information can be found in the Communication by B. M. Nestl et al. on page 253 in Issue 3, 2017 (DOI: 10.1002/cbic.201600647).