Characterization, crystallization and preliminary X - Ray diffraction analysis of an (S)-specific esterase (pfEstA) from Pseudomonas fluorescens KCTC 1767: Enantioselectivity for potential industrial applications

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) from Pseudomonas fluorescens KCTC 1767, which can be used in the production of (S)-ketoprofen, is described. Multiple sequence alignments with other hydrolases revealed that pfEstA 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 in Escherichia coli, purified to homogeneity and characterized using SDS-PAGE, MALDI-TOF MS and enantioselective analysis. pfEstA was crystallized using a solution consisting of 1 M sodium citrate, 0.1 M CHES pH 9.5, and X-ray diffraction data were collected to a resolution of 1.9 Å with an R merge of 7.9%. The crystals of pfEstA belonged to space group P212121, with unit-cell parameters a = 65.31, b = 82.13, c = 100.41 Å, = Β = = 90°.