Ene-reductases allow regio- and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene-reductase FOYE-1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel FOYE-1 to produce valuable compounds. A maximum activity of (319.7±3.2) U mg⁻¹ with NADPH or of (206.7±3.4) U mg⁻¹ with 1-benzyl-1,4-dihydronicotinamide (BNAH) for the reduction of N-methylmaleimide was observed at 30 °C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: FOYE-1 showed excellent performance in most systems with up to 20 vol% solvent and at temperatures up to 40 °C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200 mL biotransformation of 750 mg (R)-carvone afforded 495 mg of (2R,5R)-dihydrocarvone (>95 % ee), demonstrating the simplicity of handling and application of FOYE-1.

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81-84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

Ene-reductases originating from extremophiles are gaining importance in the field of biocatalysis due to higher-stability properties. The genome of the acidophilic iron-oxidizing bacterium “Ferrovum” sp. JA12 was found to harbor a thermophilic-like ene-reductase (FOYE-1). The foye-1 gene was ligated into a pET16bp expression vector system, and the enzyme was produced in Escherichia coli BL21 (DE3; pLysS) cells in yields of 10 mg L⁻¹. FOYE-1 showed remarkable activity and rates on N-phenylmaleimide and N-phenyl-2-methylmaleimide (up to 89 U mg⁻¹, >97 % conversion, 95 % (R)-selective) with both nicotinamide cofactors, NADPH and NADH. The catalytic efficiency with NADPH was 27 times higher compared to NADH. At the temperature maximum (50 °C) and pH optimum (6.5), activity was almost doubled to 160 U mg⁻¹. These findings accomplish FOYE-1 for a valuable biocatalyst in the synthesis of succinimides. The appearance of a thermophilic-like ene-reductase in an acidic habitat is discussed with respect to its phylogenetic placement and to the genomic neighborhood of the encoding gene, awarding FOYE-1 a putative involvement in a quorum-sensing process.