Characterization, crystallization and preliminary X-ray diffraction analysis of an (S)-specific esterase (pfEstA) fromPseudomonas fluorescensKCTC 1767: enantioselectivity for potential industrial applications

Abstract

The structures and reaction mechanisms of enantioselective hydrolases, which can be used in industrial applications such as biotransformations, are largely unknown. Here, the X-ray crystallographic study of a novel (S)-specific esterase (pfEstA) fromPseudomonas fluorescensKCTC 1767, which can be used in the production of (S)-ketoprofen, is described. Multiple sequence alignments with other hydrolases revealed thatpfEstA contains a conserved Ser67 within the S-X-X-K motif as well as a highly conserved Tyr156. Recombinant protein containing an N-terminal His tag was expressed inEscherichia coli, purified to homogeneity and characterized using SDS–PAGE, MALDI-TOF MS and enantioselective analysis.pfEstA was crystallized using a solution consisting of 1 Msodium citrate, 0.1 MCHES pH 9.5, and X-ray diffraction data were collected to a resolution of 1.9 Å with anRmergeof 7.9%. The crystals ofpfEstA belonged to space groupP212121, with unit-cell parametersa= 65.31,b= 82.13,c = 100.41 Å, α = β = γ = 90°.