The molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases

Abstract

Carboxylesterases containing the sequence motif GGGX catalyze hydrolysis of esters of chiral tertiary alcohols, albeit at only low to moderate enantioselectivity towards three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity towards this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this residue by alanine led to a six-fold increase of enantioselectivity (E=19) towards 2-phenyl-3-butin-2-yl acetate. However, the effect of this mutation is personalized: towards the substrate linalyl acetate, the same mutant had a reversed enantiopreference. Thus, depending on the substrate structure, the same mutant had either increased enantioselectivity or opposite enantiopreference compared to wild type enzyme.