Abstract
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.
Highlights
Chiral alcohols are one of the most important building blocks for many pharmaceutical intermediates and fine chemicals[1, 2]
Ethyl 2-oxo-4-phenylbutyrate (EOPB), which is converted to ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-EHPB)[21, 22], a key chiral intermediate for synthesis of the angiotensin converting enzyme inhibitors (ACEIs)[23], was selected as a potent substrate
The nicotinamide and ribose ring of NADP+ is oriented towards the center of the β barrel, and the adenine ring is located in the cleft between α9 and α10 with its adjacent ribose ring and diphosphate groups sandwiched by the loops connecting α8-β8 and α10-β9 (Fig. 1a)
Summary
Chiral alcohols are one of the most important building blocks for many pharmaceutical intermediates and fine chemicals[1, 2]. Based on the structural models and proposed catalytic mechanism, semi-rational engineering of Tm1743 was carried out, through which we effectively manipulated the enantioselectivity of Tm1743 to produce optically pure EHPB. Both (R)- and (S)-EHPB were obtained with ee values over 99%. In this work we illustrate the application of Tm1743 in producing chiral alcohols and demonstrate an efficient strategy to improve the enantioselectivity of a natural biocatalyst that can be applied in fine chemical and medicine synthesis
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