title: Purification and crystallization of the oxygenase component of naphthalene dioxygenase in native and selenomethionine-derivatized forms.
abstract: A new procedure was developed for the purification of the terminal oxygenase component (ISPNAP) of naphthalene dioxygenase. From a five liter culture of Escherichia coli JM109(DE3)(pDTG121), 91 mg of pure protein were obtained with a specific activity of 2.48 mumol/ min/mg protein. ISPNAP was crystallized in the rhombohedral space group R32 with cell dimensions of a = b = 179.2 A; c = 322.5 A in the hexagonal setting. The crystals are brown, indicating the presence of an intact Rieske iron-sulfur center. Problems with non-isomorphism between native data sets necessitated the preparation of a selenomethionine-substituted protein. Complete replacement of methionine with selenomethionine was achieved and the purified protein had a specific activity almost identical to native ISPNAP. Crystals from this preparation belong to the same space group and have similar cell dimensions to native ISPNAP.
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City 52242, USA.
authors.forename: K
authors.initials: K
authors.lastname: Lee
authors.forename: B
authors.initials: B
authors.lastname: Kauppi
authors.forename: R E
authors.initials: RE
authors.lastname: Parales
authors.forename: D T
authors.initials: DT
authors.lastname: Gibson
authors.forename: S
authors.initials: S
authors.lastname: Ramaswamy
mesh_terms: D018360:Crystallography, X-Ray; D049308:Dioxygenases; D014450:Electron Transport Complex III; D004926:Escherichia coli / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry*; D012645:Selenomethionine / Q000737:chemistry*
chemical_list: D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; C037989:Rieske iron-sulfur protein; D012645:Selenomethionine; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase; D014450:Electron Transport Complex III