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

Biochemical and Biophysical Research Communications
K LeeS Ramaswamy

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.

Citations

Nov 9, 2000·Letters in Applied Microbiology·M CiviliniG Tell
Mar 3, 2006·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Kyoung LeeS Ramaswamy
Jun 6, 2006·Applied and Environmental Microbiology·Sinéad M Ní ChadhainGerben J Zylstra
Feb 2, 2002·Applied and Environmental Microbiology·Daniel J LessnerDavid T Gibson
Feb 9, 2017·Journal of Chemical Information and Modeling·Diego E EscalanteAlptekin Aksan
Mar 12, 1999·Journal of Bacteriology·R E ParalesD T Gibson
Mar 14, 2000·Journal of Inorganic Biochemistry·A KarlssonS Ramaswamy

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