Abstract
Ene reductases from the Old Yellow Enzyme (OYE) family are industrially interesting enzymes for the biocatalytic asymmetric reduction of alkenes. To access both enantiomers of the target reduced products, stereocomplementary pairs of OYE enzymes are necessary, but their natural occurrence is quite limited. A library of wild type ene reductases from different sources was screened in the stereoselective reduction of a set of representative α-alkyl-β-arylenones to investigate the naturally available biodiversity. As far as the bioreduction of the ethyl ketone derivatives concerns, the results confirmed the distinctiveness of the OYE3 enzyme in affording the reduced product in the (S) configuration, while all the other tested ene reductases from the Old Yellow Enzymes family showed the same stereoselectivity toward the formation of corresponding (R) enantiomer. A possible determinant role of the “hot spot” residue in position 296 for the stereoselectivity control of these reactions was confirmed by the replacement of Phe296 of OYE1 with Ser as found in OYE3. Further investigations showed that the same stereoselectivity switch in OYE1 could be achieved also by the replacement of Trp116 with Ala and Val, these experimental results being rationalized by structural and docking studies. Moreover, an additive effect on the stereoselectivity of OYE1 was observed when coupling the selected mutations in position 296 and 116, thus providing two extremely enantioselective variants of OYE1 (W116A-F296S, W116V-F296S) showing the opposite stereoselectivity of the wild type enzyme. Lastly, the effects of the mutations on the bioreduction of carvone enantiomers were investigated as well.
Highlights
The application of enzymes, either isolated or as whole cells, as biocatalysts in organic synthesis is gaining increasing interest for the sustainable and efficient production of high-value products such as pharmaceutical intermediates and fine chemicals (Bornscheuer et al, 2012; Bornscheuer, 2018; Devine et al, 2018)
Ene reductases (ERs) from the Old Yellow Enzyme (OYE) family (EC 1.6.99.1) are flavin mononucleotide (FMN)containing oxidoreductases capable to catalyze the stereoselective reduction of α,β-unsaturated compounds activated by the presence of an electron-withdrawing group (EWG) in proximity of the C-C double bond, typically a carbonyl group or a nitro group (Gatti et al, 2013; Toogood and Scrutton, 2014, 2018; Knaus et al, 2016; Winkler et al, 2018)
Seven different ERs belonging to the OYE family and from different sources and LtB4DH, a NADPH-dependent flavin-free alkene reductase from Rattus norvegicus belonging to the medium-chain dehydrogenase superfamily (Bougioukou and Stewart, 2008), were included in the screening to improve the biocatalysts diversity
Summary
The application of enzymes, either isolated or as whole cells, as biocatalysts in organic synthesis is gaining increasing interest for the sustainable and efficient production of high-value products such as pharmaceutical intermediates and fine chemicals (Bornscheuer et al, 2012; Bornscheuer, 2018; Devine et al, 2018). Ene reductases (ERs) from the Old Yellow Enzyme (OYE) family (EC 1.6.99.1) are flavin mononucleotide (FMN)containing oxidoreductases capable to catalyze the stereoselective reduction of α,β-unsaturated compounds activated by the presence of an electron-withdrawing group (EWG) in proximity of the C-C double bond, typically a carbonyl group or a nitro group (Gatti et al, 2013; Toogood and Scrutton, 2014, 2018; Knaus et al, 2016; Winkler et al, 2018). The catalytic mechanism of OYEs is well-understood: the enzyme-bound flavin is first reduced at the expense of NAD(P)H cofactor (reductive half-reaction), a hydride is transferred from the reduced FMNH2 to the electronically activated Cβ position of the alkene substrate (Karplus et al, 1995; Toogood et al, 2010). The in situ regeneration of the reduced nicotinamide cofactor can be achieved in practical in vitro applications by using an enzymatic recycling system, e.g., that using a NAD(P)Hdependent glucose dehydrogenase (GDH) and glucose as a co-substrate
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