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 (kcat = 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.

Identification of bypass reactions leading to the formation of one central steroid degradation intermediate in metabolism of different bile salts in Pseudomonas sp. strain Chol1

“…This enzyme belongs to the old yellow enzyme family, which is known for its potential of reducing enoyl double bonds (French and Bruce, 1995;Miura et al, 1997;Williams and Bruce, 2002;Durchschein et al, 2012). This enzyme belongs to the old yellow enzyme family, which is known for its potential of reducing enoyl double bonds (French and Bruce, 1995;Miura et al, 1997;Williams and Bruce, 2002;Durchschein et al, 2012).…”

Sequential Enzymatic Conversion of α‐Angelica Lactone to γ‐Valerolactone through Hydride‐Independent C=C Bond Isomerization

“…In special cases, mostly on cyclic compounds such as α,β‐unsaturated cycloalkenones or lactones, distinct unusual catalytic behaviors of OYE homologues were observed: 1) Nicotinamide‐free dismutation catalyzed by OYE1 was identified using cyclohexen‐2‐one and 3‐oxodecalin‐4‐ene, in which one substrate molecule is first dehydrogenated by oxidized flavin, followed by reduction of a second molecule by the reduced flavin (Scheme 1 A);3 this intermolecular reaction was extended to a broad variety of substrates and OYE homologues 4. 2) Redox‐neutral C=C bond isomerization of α‐methylene‐γ‐butyrolactone to thermodynamically more stable 3‐methylfuran‐2(5  H )‐one was observed with OYE2 (Scheme 1 B); this process requires only catalytic amounts of NADH to activate the flavin by means of reduction, thereby triggering intermolecular hydride transfer from endo ‐Cβ onto exo ‐Cβ through FMN 5. In both cases, the flavin acts as hydride shuttle between two substrate molecules.…”

“…This result was surprising because reduction of C=C bonds on analogous compounds was only reported on enone derivatives so far (i.e., 1,4‐addition on activated C=C bonds). By analogy with the isomerization reaction of α‐methylene‐γ‐butyrolactone catalyzed by OYE2,5 which requires catalytic amounts of nicotinamide, the experiment was repeated with lower cofactor concentration. Upon reducing NADH concentration to substoichiometric amounts, isomerized product β‐angelica lactone ( 1 b ) could be detected, whereas consistently less saturated product 1 c was produced (Table 1).…”

“…Among all OYE homologues tested in absence of NADH, only OYE3 showed significant activity in the nicotinamide‐free C=C bond isomerization (35 % conversion in 24 h, Table 2, entry 3). Clearly, this enzymatic isomerization is distinct from the isomerization of α‐methylene‐γ‐butyrolactone by OYE2, which requires catalytic amounts of NADH cofactor to initiate hydride transfer 5. By analogy with the isomerization mechanism of Δ 5 ‐3‐ketosteroid isomerase on steroids,11 the isomerization of the unactivated C=C bond in α‐angelica lactone by OYE2 most likely relies on acid–base catalysis and is hydride independent.…”

“…[4] 2) Redox-neutralC =Cb ond isomerization of a-methylene-g-butyrolactone to thermodynamically more stable 3-methylfuran-2(5 H)one was observed with OYE2 (Scheme 1B); this process requires only catalytic amounts of NADH to activate the flavin by means of reduction,t hereby triggering intermolecular hydride transfer from endo-Cb onto exo-Cb through FMN. [5] In both cases, the flavin acts as hydride shuttle between two substrate molecules. In contrastt ot hese redox-neutral isomerization reactions,C =Cr eduction of racemic g-substituted a,b-unsaturated lactones proceeds through kinetic resolution, and dynamic kinetic resolution of a,g-disubstituted analogues wasa chieved with the OYE homologue nicotinamide-dependent cyclohexenone reductase (NCR) from Zymomonas mobilis.…”