Purification, crystallization and preliminary X-ray diffraction studies ofD-tagatose 3-epimerase fromPseudomonas cichorii

Abstract

D-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of D-psicose has not been reported with epimerases other than P. cichorii D-TE and D-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 A, beta = 102.82 degrees . Diffraction data were collected to 2.5 A resolution. The asymmetric unit is expected to contain four molecules.

Crystal Structures of d-Tagatose 3-Epimerase from Pseudomonas cichorii and Its Complexes with d-Tagatose and d-Fructose

“…13 In a previous paper, it was reported that resolution data at 2.5 Å were successfully collected from a crystal of P. cichorii D-TE grown by the vapor diffusion method using a protein solution (3.5 mg/ ml P. cichorii D-TE in 20 mM Tris-HCl, pH 8.0) and a reservoir solution (20% (w/v) polyethylene glycol 10,000; 0.1 M Hepes buffer, pH 7.5; 0.01 M strontium chloride).…”

“…12 Crystallization and preliminary X-ray analyses of P. cichorii D-TE were already reported. 13 Here we report the crystal structures of P. cichorii D-TE alone and in complexes with D-tagatose (P. cichorii D-TE/D-tagatose) and D-fructose (P. cichorii D-TE/ D-fructose).…”

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109

“…At present, and for the first time, the highly purified DTE protein has been obtained from E. coli. The DTE protein could be dimeric enzyme, because D-tagatose 3-epimerase (D-TE) from P. cichorii (290 amino acid residues, 32 615 Da, 65 kDa dimeric enzyme) catalyzes the epimerization of various ketohexoses at the C3 position [2,15]. The predicted pI of the DTE protein from E. coli is 5.31.…”

“…D-Tagatose 3-epimerase from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position [11,12]. For this study, the D-tagatose 3-epimerase (DTE) gene in Escherichia coli was cloned and inserted into the plasmid vector pET-15b, the recombinant plasmid (pET-15b-DTE) was transformed into the strain BL21 (DE3), and through isopropyl b-D-1-thiogalactopyranoside (IPTG) induction a large quantity of target protein DTE is produced.…”

Recent advances on applications and biotechnological production of d-psicose

“…The three-dimensional structures of A. tumefaciens DPEase (Kim et al 2006b and2006c) and P. cichorii DTEase (Yoshida et al 2007a and2007b) were already solved, and the active sites were well identified. The enzyme contains a metal ion binding site, and it is suggested that the metal ion plays a pivotal role in catalysis by anchoring the bound substrate of D-fructose.…”