Aromatic interactions specific to aryl radicals were introduced into two esterases, BioH from Escherichia coli and RspE from Rhodobacter sphaeroides to control their enantioselectivity in the asymmetric hydrolysis of prochiral aryl glutaric acid diesters. As a result, the enantiomeric excess (ee) of the S-product of dimethyl 3-phenylglutarate was increased from 25% (BioH wild type) to 96% (B_L83F/L86F) and from 13% (RspE wild type) to >99% (R_Y27R), respectively, while another variant of RspE R_M121F gave a reversed ee of 50% (R-product). Similar enhancement or reversion of enantioselectivity were also observed in the hydrolysis of three other prochiral aryl diesters (dimethyl 3-(4-flouro)-phenylglutarate, dimethyl 3-(4-cholo)-phenylglutarate and dimethyl 3-(3,4-dicholo)-phenylglutarate). Especially, the mutant R_Y27R was shown to be an excellent S-selective hydrolase for prochiral aryl diesters, with ee of all S-products >99%. In the mutants with altered enantioselectivity, the successful introduction of designed aromatic interactions was confirmed by molecular dynamics simulations and binding free energy analysis. These results demonstrate that aromatic interaction is one of the origins of enzyme enantioselectivity, the tuning of which leads to dramatic change in enantioselectivity. Besides, the successful engineering of the enantioselectivity in two different proteins toward four different substrates suggests that the introduction of aromatic interactions is a generally applicable strategy in the control of enantioselectivity toward aryl substrates.
Keywords: aromatic interactions; enantioselectivity; esterase; molecular dynamics simulation; rational design.
© 2014 Wiley Periodicals, Inc.