title: Biochemical characterisation of a NADPH-dependent carbonyl reductase from Neurospora crassa reducing α- and β-keto esters.
abstract: A gene encoding an NADPH-dependent carbonyl reductase from Neurospora crassa (nccr) was cloned and heterologously expressed in Escherichia coli. The enzyme (NcCR) was purified and biochemically characterised. NcCR exhibited a restricted substrate spectrum towards various ketones, and the highest activity (468U/mg) was observed with dihydroxyacetone. However, NcCR proved to be very selective in the reduction of different α- and β-keto esters. Several compounds were converted to the corresponding hydroxy ester in high enantiomeric excess (ee) at high conversion rates. The enantioselectivity of NcCR for the reduction of ethyl 4-chloro-3-oxobutanoate showed a strong dependence on temperature. This effect was studied in detail, revealing that the ee could be substantially increased by decreasing the temperature from 40 °C (78.8%) to -3 °C (98.0%). When the experimental conditions were optimised to improve the optical purity of the product, (S)-4-chloro-3-hydroxybutanoate (ee 98.0%) was successfully produced on a 300 mg (1.8 mmol) scale using NcCR at -3 °C.
authors: evocatal GmbH, Merowingerplatz 1a, 40225 Düsseldorf, Germany.
authors: Nina
authors: N
authors: Richter
authors: Werner
authors: W
authors: Hummel
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D004952:Esters / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004952:Esters; D005656:Fungal Proteins; D007659:Ketones; D011993:Recombinant Fusion Proteins; D009249:NADP; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase