title: An ene reductase from Clavispora lusitaniae for asymmetric reduction of activated alkenes.
abstract: A putative ene reductase gene from Clavispora lusitaniae was heterologously overexpressed in Escherichia coli, and the encoded protein (ClER) 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 α,β-unsaturated carboxylic esters. The purified ClER exhibited a relatively high activity of 7.3 U mg(prot)⁻¹ for ketoisophorone while a remarkable catalytic efficiency (k(cat)/K(m)=810 s⁻¹ mM⁻¹) was obtained for 2-methyl-cinnamaldehyde due to the high affinity. A series of prochiral activated alkenes were stereoselectively reduced by ClER furnishing the corresponding saturated products in up to 99% ee. The practical applicability of ClER was further evaluated for the production of (R)-levodione, a valuable chiral compound, from ketoisophorone. Using the crude enzyme of ClER and glucose dehydrogenase (GDH), 500 mM of ketoisophorone was efficiently converted to (R)-levodione with excellent stereoselectivity (98% ee) within 1h. All these positive features demonstrate a high synthetic potential of ClER in the asymmetric reduction of activated alkenes.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ni
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Hui-Lei
authors.initials: HL
authors.lastname: Yu
authors.affiliation: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: jianhexu@ecust.edu.cn.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D000475:Alkenes / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D001711:Biotransformation; D003001:Cloning, Molecular; D000081005:Cyclohexane Monoterpenes; D003512:Cyclohexanones / Q000302:isolation & purification / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D055163:Metschnikowia / Q000201:enzymology* / Q000235:genetics; D015394:Molecular Structure; D039821:Monoterpenes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000475:Alkenes; D000081005:Cyclohexane Monoterpenes; D003512:Cyclohexanones; D005420:Flavoproteins; D005656:Fungal Proteins; D039821:Monoterpenes; D011993:Recombinant Fusion Proteins; C494596:levodione; C476259:ketoisophorone; D009249:NADP; C006923:carvone; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases
keywords: Activated alkenes; Asymmetric reduction; Biocatalysis; Old yellow enzyme; ene Reductase