Semi-Rational Engineering a Carbonyl Reductase for the Enantioselective Reduction of beta-Amino Ketones

Chiral 3-(dimethylamino)-1-phenylpropan-1-ol (2a) and 3-(dimethylamino)-1-(2-thienyl)-1-ol (2b) are very important intermediates for the synthesis of antidepressants. The mutant M242F/Q245T of a carbonyl reductase (SSCR) from Sporobolomyces salmonicolor AKU4429 catalyzed the reduction of 3-(dimethylamino)-1-phenylpropan-1-one (la) to the (S)-2a (28% ee). The combinatorial active-site saturation of this enzyme resulted in two mutants P170R/L174Y and P170H/L174Y, which catalyzed the reduction of la and 3-(dimethylamino)-1-(2-thienyl)-propan-1-one (1b) to give the (R)-gamma-amino alcohols with up to 95% ee, respectively. The individual site saturation mutagenesis of Pro170 and Leu174 revealed that Pro170 did not significantly affect the enzyme enantioselectivity toward la and 1b, whereas residue Leu174 played a critical role in determining the enantioselectivity. Mutant L174W catalyzed the reduction of la to the (S)-gamma-amino alcohol with increased enantioselectivity from 28% ee to 96% ee, although mutant L174Y exhibited (R)-preference in 88% ee. For 1b, the (R)-alcohol was obtained with 95% ee by using variant L174Y as the catalyst, whereas L174W exhibited (S)-preference in 65% ee. The kinetic studies indicated that catalytic efficiencies (k(cat)/K-m) of these mutants were also improved. The enzyme substrate docking provided some insights into the structural basis for the reversal of enantioselectivity by the substitution of Leu174.