Structural Basis for the Enantioselectivity of Esterase Est-Y29 toward (S)-Ketoprofen

Abstract

The thermostable esterase Est-Y29, belonging to the family VIII lipolytic esterases isolated from metagenomes extracted from the topsoil in Republic of Korea, was identified as a promising catalyst for the production of (S)-ketoprofen, an important nonsteroidal anti-inflammatory drug (NSAID). For industrial applications, the enantioselectivity of the enzyme toward the S-enantiomer of the racemic ketoprofen ester substrate needs to be improved. To understand the structural basis of Est-Y29 enantioselectivity, which is necessary for the rational design of an enzyme with enhanced enantioselectivity, we solved the crystal structures of Est-Y29 bound to (S)-ketoprofen at 1.69 angstrom resolution. Structural analyses revealed that the S-enantiomer can be stabilized by a pi-interaction between the methyl substituent at the chiral carbon of the ligand and the aromatic pocket formed by Tyr123, Phe125, and Tyr170. This finding is further supported by the highly improved enantioselectivity of the mutant Est-Y29 (F125W) toward (S)-ketoprofen due to the enhanced pi-interaction. Our results provide the molecular basis of the enantioselectivity of Est-Y29 against (S)-ketoprofen and further offer the opportunity for the rational design of enzyme enantioselectivity as well as potential applications of the mutant Est-Y29 to industrial biocatalysts.