Biochemical characterisation of a NADPH-dependent carbonyl reductase from Neurospora crassa reducing α- and β-keto esters.

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.