Overexpression, purification, characterization and preliminary crystallographic study of phosphoglycolate phosphatase from Shigella flexneri 2a strain 301.
Phosphoglycolate phosphatase has a salvage function in the metabolism of the 2-phosphoglycolate formed during bacterial DNA repair. In order to better understand its dimerization behaviour, the influence of metal ions on its activity and its catalytic mechanism at the molecular level, recombinant phosphoglycolate phosphatase from Shigella flexneri was overexpressed, purified, characterized and crystallized by the hanging-drop vapour-diffusion method at 291 K using polyethylene glycol 3500 as a precipitant and zinc acetate as an additive. The crystals belonged to space group R3, with unit-cell parameters a = 88.1, b = 88.1, c = 259.2 A, corresponding to the presence of two molecules in the asymmetric unit. SeMet-labelled protein was also prepared and crystallized for use in phase determination. Initial structure determination using the multiwavelength anomalous dispersion (MAD) method clearly revealed that SfPGPase bears an alpha-helical cap domain that differs from that of a previously reported orthologue.
Liu
Heli
H
Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.
Zhou
Huina
H
Zhu
Deyu
D
Bi
Ruchang
R
D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism; D004058:Diffusion; D007477:Ions; D008027:Light; D008670:Metals / Q000737:chemistry; D015233:Models, Statistical; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012763:Shigella flexneri / Q000201:enzymology*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C022408:phosphoglycolate phosphatase; D010744:Phosphoric Monoester Hydrolases