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

Thermostable esterase Est-Y29, a family VIII lipolytic esterase isolated from metagenomes, was identified as a promising catalyst for the production of (S)-ketoprofen, an important member of nonsteroidal anti-inflammatory drugs (NSAIDs). However, for the industrial application, its enantioselectivity towards the S-enantiomer of the racemic ketoprofen ester substrate need to be improved. In this study, we solved the crystal structures of the wild-type and the mutant Est-Y29 bound to the (S)-ketoprofen. We confirmed that S-enantiomer can be stabilized by a hydrophobic interaction between the methyl substituent at the chiral carbon of the ligand and the hydrophobic dent formed by Tyr123, Phe125 and Tyr170. Supportively, an Est-Y29 mutant F125W showed the highly improved (S)-ketoprofen enantioselectivity, possibly due to enhanced hydrophobic interaction between the (S) enantiomer and Est-Y29 mutant. 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 the enzyme enantioselectivity.