RESUMEN
A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydroxy-4-phenylbutyrate ((S)-EHPB), which is less desirable, is preferred with an enantiomeric excess (ee) value of 76.5%. Thus, altering the enantioselectivity of Tm1743 to obtain the more valuable product (R)-EHPB for angiotensin drug synthesis is highly desired. In this work, we determined the crystal structure of Tm1743 in complex with its cofactor NADP+ at 2.0 Å resolution, and investigated the enantioselectivity of Tm1743 through semi-rational enzyme design. Molecular simulations based on the crystal structure obtained two binding models representing the pro-S and pro-R conformations of EOPB. Saturation mutagenesis studies revealed that Trp21 and Trp86 play important roles in determining the enantioselectivity of Tm1743. The best (R)- and (S)-EHPB preferring Tm1743 mutants, denoted as W21S/W86E and W21L/W118H, were identified; their ee values are 99.4% and 99.6% and the catalytic efficiencies are 0.81 and 0.12 mM-1s-1, respectively. Our work presents an efficient strategy to improve the enantioselectivity of a natural biocatalyst, which will serve as a guide for further exploration of new green catalysts for asymmetric reactions.
