Purification, crystallization and preliminary X-ray analysis of the Escherichia coli glucose-1-phosphatase.
Encoded by the agp gene, Escherichia coli glucose-1-phosphatase hydrolyzes glucose-1-phosphate in the periplasmic space of the bacterium. It is a potential drug-design target because inositol phosphatases have been identified as important virulence determinants in several human and animal pathogens. The enzyme was isolated and purified to homogeneity from a strain of E. coli CU1867 (an appA-deficient mutant). Crystals were obtained overnight by the equilibrium vapour-diffusion method from a solution containing 10 mg ml(-1) enzyme, 1.2 M ammonium sulfate and 25% polyethylene glycol monomethyl ether 5000 in 0.1 M MES at pH 6.5. The crystals belong to space group R3, with unit-cell parameters a = b = 156.0, c = 92.2 A. The diffraction limit was 2.6 A at a rotating-anode X-ray source; a 2.7 A resolution data set has been collected using light mineral oil as a cryoprotectant. The data set was 95.2% complete, with an R(sym) of 0.058. There were two monomers of glucose-1-phosphatase in the asymmetric unit, which correspond to a V(M) of 2.36 A Da(-1) and 47.5% solvent content. Self-rotation analysis unambiguously shows a twofold non-crystallographic symmetry.
Jia
Z
Z
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada. jia@post.queensu.ca
Cottrill
M
M
Pal
G P
GP
Lee
D
D
Sung
M
M
Forsberg
C W
CW
Phillips
J P
JP
D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D014961:X-Ray Diffraction
D029968:Escherichia coli Proteins; C062887:agp protein, E coli; D010744:Phosphoric Monoester Hydrolases