Structure-Based Insight into the Asymmetric Bioreduction of the C=C Double Bond of alpha,beta-Unsaturated Nitroalkenes by Pentaerythritol Tetranitrate Reductase

Abstract

Biocatalytic reduction of alpha- or beta-alkyl-beta-arylnitroalkenes provides a convenient and efficient method to prepare chiral substituted nitroalkanes. Pentaerythritol tetranitrate reductase (PETN reductase) from Enterobacter cloacae st. PB2 catalyses the reduction of nitroolefins such as 1-nitrocyclohexene (1) with steady state and rapid reaction kinetics comparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryl-1-nitropropenes (4a-d) to their equivalent (S)-nitropropanes 9a-d. The enzyme shows a preference for the (Z)-isomer of substrates 4a-d, providing almost pure enantiomeric products 9a-d (ees up to > 99%) in quantitative yield, whereas the respective (E)-isomers are reduced with lower enantioselectivity (63-89% ee) and lower product yields. 1-Aryl-2-nitropropenes (5a, b) are also reduced efficiently, but the products (R)-10 have lower optical purities. The structure of the enzyme complex with 1-nitrocyclohexene (1) was determined by X-ray crystallography, revealing two substrate-binding modes, with only one compatible with hydride transfer. Models of nitropropenes 4 and 5 in the active site of PETN reductase predicted that the enantioselectivity of the reaction was dependent on the orientation of binding of the (E)- and (Z)-substrates. This work provides a structural basis for understanding the mechanism of asymmetric bioreduction of nitroalkenes by PETN reductase.

Scheme 1

(a) General mechanism of the biocatalytic reduction of α,β-unsaturated alkenes catalyzed by the OYE family. (b) General mechanism of the biocatalytic reduction of α,β-unsaturated nitroalkenes involving nitronate formation and aci-nitro tautomerisation of the product formed. EWG=electron withdrawing group; *=chiral centre.

Scheme 2

Substrates and products of C=C double bond bioreduction catalysed by PETN reductase.

Scheme 3

Asymmetric bioreduction of α,β-disubstituted nitroalkenes by OYE enzymes. OYE1-3=old yellow enzymes 1-3; NCR=NCR enoate reductase from Zymomonas mobilis; OPR1 and 3=12-oxophytodienoate reductase isoforms from Lycopersicon esculentum; YqjM=from Bacillus subtilis.[8-11]

Scheme 4

Theoretical model for the selectivity of PETN reductase with (E)-4 (a) and (E)-5 (b), adapted from[45]. AA=amino acid residue/s modelled to interact with the substrate.

Figure 1

Spectral changes of nitroalkene reduction by PETN reductase under aqueous conditions. Inset: Time course of absorbance changes upon substrate reduction. The arrows indicate the direction of spectral change. (a) (Z)-2-(4′-chlorophenyl)-1-nitropropene [(Z)-4c]; (b) (E)-2-(4′-chlorophenyl)-1-nitropropene [(E)-4c]; (c) (Z)-1-(4′-chlorophenyl)-2-nitropropene [(Z)-5b]; (d) (E)-1-(4′-chlorophenyl)-2-nitropropene [(E)-5b].

Figure 2

Time course of the PETN reductase-catalysed reduction of (Z)- and (E)-isomers of 2-(4′-chlorophenyl)-1-nitropropene 4c (a and b, respectively) under biphasic conditions. Substrates: □=(Z)-4c; ○=(E)-4c; product: ▲=(R)-9c. Time course of the PETNR-catalysed reduction of (Z)- and (E)-isomers of 1-(4′-chlorophenyl)-2-nitropropene 5b (c and d, respectively) under biphasic conditions. Substrates: □=(Z)-5b; ○=(E)-5b; product: ▲=(R)-10b. The % ees throughout the reactions were fairly consistent with that of the final product (% ee was 52-60% and 53-56% for (Z)-5a and (E)-5a, respectively) and dropped by only 1% per day.

Figure 3

Active site of PETN reductase containing bound substrate. The figures were generated in Pymol.[56] All residues are shown as atom-coloured sticks with green and yellow carbons for protein and FMN, respectively. Hydrogen bonds are indicated by dotted lines (grey lines indicate the bond is behind the side chain). (a) Stereo view of the 2 |F_o|–|F_c| electron density map contoured at 1 σ and final atomic model of the active site of 1-bound PETN reductase. The catalytic and non-productive positions for 1 are shown as atom-coloured sticks with pink carbons and grey lines, respectively. Dual models of the (b) (E)- and (c) (Z)-isomers of 4c and 5b bound in the active site of PETN reductase. The substrates (E)- and (Z)-4c are shown as atom-coloured sticks with magenta and blue carbons for models 1 and 2, respectively. The substrates (E)- and (Z)-5b are shown as atom-coloured lines with magenta and blue carbons for models 1 and 2, respectively. The hydrogen bonds are colour-coded to indicate the substrate and model, with large and small dots for (E/Z)-4c and (E/Z)-5b substrates, respectively.