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 coinparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryi-1-nitropropenes (4ad) 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)-isorners 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. ; Toogood, Helen S. Fryszkowska, Anna Hare, Victoria Fisher, Karl Roujeinikova, Anna Leys, David Gardiner, John M. Stephens, Gill M. Scrutton, Nigel S.