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 oxy genase component (ISPNAP) of naphthalene dioxygenase. From a five Lite r culture of Escherichia coli JM109(DE3)(pDTG121), 91 mg of pure prote in were obtained with a specific activity of 2.48 mu mol/min/mg protei n, ISPNAP was crystallized in the rhombohedral space group R32 with ce ll dimensions of a=b=179.2 Angstrom; c=322.5 Angstrom in the hexagonal setting, The crystals are brown, indicating the presence of an intact Rieske iron-sulfur center. Problems with non-isomorphism between nati ve data sets necessitated the preparation of a selenomethionine substi tuted protein, Complete replacement of methionine with selenomethionin e was achieved and the purified protein had a specific activity almost identical to native ISPNAP Crystals from this preparation belong to t he same space group and have similar cell dimensions to native ISPNAP. (C) 1997 Academic Press.