Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase fromBifidobacterium breve

Abstract

Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapourdiffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl 2 , 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å , and diffracted to beyond 1.7 Å resolution.

Unique Sugar Metabolic Pathways of Bifidobacteria

“…[122][123][124] Very recently, crystallization of phosphoketolase from Lactococcus lactis 125) and structure determination of a substrate-free form of XFPK from B. longum JCM1217 126) were reported. Our group also succeeded in crystallization 127) and structure determination 128) of XFPK from B. breve (Fig. 6B).…”

Crystal Structures of Phosphoketolase

“…However, they share very low sequence homology, with a sequence identity of 15% based on structural alignment with TK from Saccharomyces cerevisiae (ScTK). Three active peaks of BbXFPK appeared on gel filtration chromatography with different estimated sizes (24). A sample from the major homohexameric peak successfully crystallized, whereas those from the two minor peaks (homodimer and homotetramer) failed to crystallize.…”

“…Enzyme Preparation and Assay-Construction of the expression vector, protein expression, purification, and kinetic analysis employing F6P as the donor substrate were performed as described previously (24). Mutants of BbXFPK were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the primers summarized in supplemental Table 2.…”

“…Crystallography-Native (resting) BbXFPK and its mutants were crystallized according to the method described previously (24) were obtained by cocrystallization with 5 mM potassium phosphate. The mother liquor containing 20% ethylene glycol was used as a cryoprotectant, except for the BbXFPK/P i complex for which 20% glycerol was used.…”

“…Recently, preliminary x-ray crystallographic studies of XPK from Lactococcus lactis (23) and XFPK from Bifidobacterium breve 203 (BbXFPK) (24) are reported. In this study we report the crystal structure of BbXFPK as the first three-dimensional structure of PK.…”