Open AccessCharacterization, crystallization and preliminary X‐ray diffraction analysis of an ( S )‐specific esterase ( pf EstA) from Pseudomonas fluorescens KCTC 1767: enantioselectivity for potential industrial applications
Author(s) -
Kim Seulgi,
Duc Ngo Tri,
Kim Kyeong Kyu,
Kim T. Doohun
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112040626
Subject(s) - pseudomonas fluorescens , esterase , escherichia coli , crystallization , chemistry , crystallography , pseudomonas , stereochemistry , enzyme , biochemistry , bacteria , biology , organic chemistry , genetics , gene
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 ( pf EstA) 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 pf EstA 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. pf EstA 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 pf EstA belonged to space group P 2 1 2 1 2 1 , with unit‐cell parameters a = 65.31, b = 82.13, c = 100.41 Å, α = β = γ = 90°.