Purification and Characterization of an Enone Reductase from Sporidiobolus salmonicolor TPU 2001 Reacting with Large Monocyclic Enones

Abstract

We discovered a novel enone reductase from Sporidiobolus salmonicolor TPU 2001 (SsERD) and expressed the gene in Escherichia coli. The enzyme catalyzed the reduction of (E)‐3‐methylcyclopentadec‐2‐en‐1‐one (E‐2), cyclopentadec‐2‐en‐1‐one (3), and cyclododec‐2‐en‐1‐one (4) to (S)‐muscone (S‐1), cyclopentadecan‐1‐one (5), and cyclododecan‐1‐one (6), respectively. The apparent Km and Vmax values for E‐2 were estimated to be 4.9±0.4 μm and 100±1.4 nmol min−1 mg−1, respectively. The enzyme was specific to NADPH, and cysteine residues strongly affected the enzyme activity. The enzyme exhibited the highest activity at pH 8.0 and high stability in the pH range from 4.5 to 11.0. Using 10 mU of the enzyme, S‐1 was synthesized from 0.1 mm E‐2 with 94.8 % yield and 100 % enantiomeric excess by incubation at pH 7.0 and 30 °C for 60 min. We further successfully constructed an enone reductase with high specific activity by mutation of SsERD. The Y67A variant from SsERD exhibited 4.5 times higher specific activity and 3 times higher catalytic efficiency toward E‐2. This is the first report of an enzyme catalyzing the reduction of carbon–carbon double bond of large monocyclic enones.

Catalytic Asymmetric Conjugate Reduction

“…[200,212] Larger cyclic enones are also suitable substrates, as in the case of SsERD from Sporidiobolus salmonicolor TPU 2001, which reduced a 16-membered cyclic enone to render unnatural (S)-muscone in 95 % yield with > 99 % ee. [215] Bioreduction of 4-ketoisophorone to levodione 103 a, a useful starting material in the synthesis of carotenoids, is a benchmark reaction for which at least 11 different OYE family members are known to produce the R enantiomer with varying enantioselectivity (OYE1-3, NCR, ClER, OPR1, OPR3, YqjM, PETNR from Enterobacer cloacae, TOYE). [178,186,190,191,197,200,214,216,217] ee values higher that 95 % have been reported for OYE1-OYE3, NCR, and ClER.…”

Expanding the Biocatalytic Toolbox with a New Type of ene/yne‐Reductase from Cyclocybe aegerita

“…Other ene-reductases from the MDR-superfamily as NtDBR (> 1,600-fold) or SsERD (no activity with NADH) also prefer NADPH as hydride donor. [23,24] This is not surprising, due to the fact that these enzymes share several amino acids, such as G189, K193 and Y208, which were shown to be involved in NADPH binding by the crystal structures of NtDBR and AtDBR (Supporting information Figure S1A, B). [4,20]…”

“…So far, only one study targeted an ER from the yeast-like Basidiomycota Sporidiobolus salmonicolor that reduced unnatural large monocyclic enones like (E)-3-methylcyclopentadec-2-en-1-one, cyclopentadec-2-en-1-one, and cyclododec-2-en-1-one to their corresponding saturated ketones. [23] Hence, this study investigated the biocatalytic value of the first ER from a filamentous fungi of the phylum Basidiomycota as a member of the MDR-superfamily. This ER turned out to exhibit novel biocatalytic activities displaying a valuable and versatile biocatalyst with high potential for future approaches.…”

Introduction of Chiral Centers to α- and/or β-Positions of Carbonyl Groups by Biocatalytic Asymmetric Reduction of α,β-Unsaturated Carbonyl Compounds

“…Entry 8 shows that the ene reductase from S. salmonicolor TPU 2001 furnished unnatural ( S )-( + )-muscone in 95% yield and 100% ee without adding glucose and GDH. 25 The catalyst was specific for the reduction of ( E )-3-methyl-2-cyclopentadecenone, but not active for the ( Z )-enone, along with enones of smaller ring size. Development of a new catalytic system to provide the precious natural ( R )-(–)-muscone ( 2 ) is expected.…”

“…The (Z )-double bond isomer was not reduced. Catalytic hydrogenation of the product led to the γ 2 -aminoester (S)-pregabalin (25), an analogue of the neurotransmitter GABA (Figure 6).…”