Unusual C=C bond isomerization of an α,β-unsaturated γ-butyrolactone catalysed by flavoproteins from the old yellow enzyme family

Abstract

An unexpected, redox-neutral C=C bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (k(cat) =0.076 s(-1) ) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN-mediated hydride addition onto exo-Cβ, followed by hydride abstraction from endo-Cβ', which is in line with the well-established C=C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene-reductases.

Scheme 1

Biocatalytic C=C bond reduction and isomerization of α,β-unsaturated γ-butyrolactones 1 a and 2 a.

Scheme 2

Proposed mechanism of C=C bond isomerization from isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) catalysed by isopentenyl diphosphate isomerases type 2 (IDIs-2).[19, 34] B = N5 of FMNH₂, A = N5-H of FMNH₂ or N5-H₂⁺ of protonated flavin, PP = diphosphate.

Figure 1

Time courses of C=C isomerization and of reduction of α-methylene-γ-butyrolactone (1 a), both catalysed by OYE2. Substrate 1 a: ○. Isomerization product 2 a: •. Reduction product (R)-1 b: ▪ (conversion) and × (ee). Final end-point data after 26 h were 78 % 2 a and 22 % (R)-1 b (78 % ee). Reaction conditions: substrate (10 mm), ene-reductase OYE2 (100 μg mL⁻¹), NADH (15 mm), Tris⋅HCl buffer (50 mm, pH 7.5), 30 °C, 120 rpm.

Scheme 3

Proposed mechanism for C=C bond isomerization through hydride migration (red) versus C=C bond reduction (blue).