RECOMBINANT ESCHERICHIA-COLI STRAINS SYNTHESIZE ACTIVE FORMS OF NAPHTHALENE DIOXYGENASE AND ITS INDIVIDUAL ALPHA-SUBUNIT AND BETA-SUBUNIT

Pseudomonas sp. strain NCIB 9816-4 utilizes naphthalene dioxygenase (N DO), a multicomponent enzyme system, to initiate naphthalene degradati on. The terminal component of NDO is an iron-sulfur protein (ISPNAP) w ith an alpha(2) beta(2) subunit composition. The structural genes enco ding the alpha (nahAc) and beta (nahAd) subunits were cloned separatel y and together into expression vectors where transcription is under th e control of the T7 promoter. The recombinant plasmids were transforme d into Escherichia coli JM109[pGP1-2] and the synthesis of ISPNAP and its alpha and beta subunits was determined by SDS-PAGE. Low expression of nahAd was shown to be due to inefficient initiation of translation , but a sixfold increase in the amount of beta subunit synthesized was achieved in a coupled translation system. Inclusion bodies were found in all recombinants. Increased levels of soluble active proteins were obtained when E. coli JM109(DE3), used as the host strain for recombi nant plasmid, was grown at 25 degrees C. ISPNAP from JM109(DE3)[pDTG12 1] was purified to homogeneity and shown to have the same properties a s those determined for the enzyme purified from NCIB 9816-4. Active IS PNAP was also obtained by mixing cell extracts from separate strains t hat synthesized the alpha and beta subunits. The availability of large amounts of purified ISPNAP and its a and P subunits will facilitate f uture studies on the mechanism of oxygen fixation by NDO.