title: Characterization, crystallization and preliminary X-ray diffraction analysis of an (S)-specific esterase (pfEstA) from Pseudomonas fluorescens KCTC 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) 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 Rmerge of 7.9%. The crystals of pfEstA belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=65.31, b=82.13, c=100.41 Å, α=β=γ=90°.
authors.affiliation: Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon 443-749, Republic of Korea.
authors.forename: Seulgi
authors.initials: S
authors.lastname: Kim
authors.forename: Tri Duc
authors.initials: TD
authors.lastname: Ngo
authors.forename: Kyeong Kyu
authors.initials: KK
authors.lastname: Kim
authors.forename: T Doohun
authors.initials: TD
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D016384:Consensus Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D004950:Esterases / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004950:Esterases