Purification and crystallization of the oxygenase component of naphthalene dioxygenase in native and selenomethionine-derivatized forms.

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.