A putative ene reductase gene from Clavispora lusitaniae was heterologously overexpressed in Escherichia coli, and the encoded protein (CIER) was purified and characterized for its biocatalytic properties. This NADPH-dependent flavoprotein was identified with reduction activities toward a diverse range of activated alkenes including conjugated enones, enals, maleimide derivative and alpha, beta-unsaturated carboxylic esters. The purified CIER exhibited a relatively high activity of 7.3 U mg(prot)(-1) for ketoisophorone while a remarkable catalytic efficiency (k(cat)/K-m, = 810s(-1) mM(-1)) was obtained for 2-methyl-cinnamaldehyde due to the high affinity. A series of prochiral activated alkenes were stereoselectively reduced by CIER furnishing the corresponding saturated products in up to 99% ee. The practical applicability of CIER was further evaluated for the production of (R)-levodione, a valuable chiral compound, from ketoisophorone. Using the crude enzyme of CIER and glucose dehydrogenase (GDH), 500 mM of ketoisophorone was efficiently converted to (R)-levodione with excellent stereoselectivity (98% ee) within 1 h. All these positive features demonstrate a high synthetic potential of CIER in the asymmetric reduction of activated alkenes. (c) 2014 Elsevier Inc. All rights reserved.