A recombinant carboxylesterase (rPPE) from Pseudomonas putida ECU1011 was previously cloned and engineered to give a potential application for resolving chiral alpha-hydroxy acids including mandelic acids and derivatives. Two variants rPPE(w187H) and rPPE(w287A) showed a similar to 100-fold increase in activity towards rac-2-acetoxy-2-(2 '-chlorophenyl) acetate (rac-AcO-CPA), but rPPE(D287A) had a significant decrease in enantioselectivity (E = 8.7) compared to rPPE(w187H),4 and the wild-type rPPE (rPPE(wr)) (E > 200). Here we report the crystal structures of rPPEwr and rPPE(w187H), both by themselves and in complex with the substrate, to elucidate the structural basis of this phenomenon. An inactive mutation of nucleophile residue S159A was introduced to obtain the structure of rPPE(S159A/W187H) complexed with (S)-AcO-CPA. The structural analysis reveals that the side chain of residue Asp287 in rPPE(WT) would have a potential steric conflict with (S)-AcO-CPA when the substrate binds at the active site of the enzyme. However, the mutation W187H could facilitate the relocation of Asp287, while D287A directly eliminates the hindrance of Asp287, both of which offer sufficient space for the binding and hydrolysis of substrate. Moreover, Asp287 generates one site of the "three-point attachment model" as a hydrogen-bond donor that determines the excellent enantioselectivity of rPPE in chiral recognition, and D287A would obviously destroy the hydrogen bond and result in the low enantioselectivity of rPPE(D287A). (C) 2014 Elsevier Inc. All rights reserved.