Asymmetric Reduction of (R)-Carvone through a Thermostable and Organic-Solvent-Tolerant Ene-Reductase

Abstract

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.

Keywords: Old Yellow Enzymes; biocatalysis; biotransformations; cofactor mimics; oxidoreductases; solvent stability.

Scheme 1

Stereoselective reduction of (R)‐carvone through the action of an ene‐reductase (ER). The nicotinamide (biological or synthetic, NA) acts as an electron donor to reduce the flavin cofactor FMN of the ER; this subsequently allows the transfer of a hydride to C_α of the unsaturated substrate. A proton from a conserved Tyr residue in the ER is added to C_β to yield (2R,5R)‐dihydrocarvone.2, 6

Figure 1

Michaelis–Menten kinetic analysis of FOYE‐1 with BNAH as cosubstrate. The standard enzyme assay was performed as described in the Experimental Section, in KH₂PO₄/Na₂HPO₄ buffer at 22.5 °C. FOYE‐1 (8.6 nm, 0.375 μg mL⁻¹, holoprotein) was used without the addition of extra FMN. The concentration of A) BNAH, or B) substrate 2 was varied (BNAH 0–1200 μm, 2 0–200 μm), while the other was kept in excess. Data were analyzed by nonlinear fitting of the Michaelis–Menten equation with the aid of the KaleidaGraph software package (Table 3).

Figure 2

FOYE‐1 activity in presence of cosolvents. The standard enzyme assay was performed while the concentrations of solvents were varied, initial rates were determined. A) NADPH (200 μm), or B) BNAH (1000 μm) served as electron donor; N‐methylmaleimide (2, 1 mm) was used as substrate. Data are shown as values relative to an enzyme assay without cosolvents [A) 100 %=140 U mg⁻¹, B) 100 %=170 U mg⁻¹].

Figure 3

Biotransformation of (R)‐carvone (12) through the action of FOYE‐1 with different protein preparations, 10 mL scale. For comparison, A) a chromatographically enriched, or B) a crude extract preparation was employed; 1 μm enzyme was applied to convert 5 mm (7.5 mg) substrate 12 while the electron donor BNAH was fed stepwise (initial 10 mm+7.5 mm h⁻¹ in solid form). Substrate and products were analyzed by chiral GC.