Recombinant Escherichia coli strains synthesize active forms of naphthalene dioxygenase and its individual alpha and beta subunits.
Pseudomonas sp. strain NCIB 9816-4 utilizes naphthalene dioxygenase (NDO), a multicomponent enzyme system, to initiate naphthalene degradation. The terminal component of NDO is an iron-sulfur protein (ISPNAP) with an alpha 2 beta 2 subunit composition. The structural genes encoding the alpha (nahAc) and beta (nahAd) subunits were cloned separately and together into expression vectors where transcription is under the control of the T7 promoter. The recombinant plasmids were transformed 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 recombinant plasmid, was grown at 25 degrees C. ISPNAP from JM109(DE3)[pDTG121] was purified to homogeneity and shown to have the same properties as those determined for the enzyme purified from NCIB 9816-4. Active ISPNAP was also obtained by mixing cell extracts from separate strains that synthesized the alpha and beta subunits. The availability of large amounts of purified ISPNAP and its alpha and beta subunits will facilitate future studies on the mechanism of oxygen fixation by NDO.
Suen
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WC
Department of Microbiology, University of Iowa, Iowa City 52242.
Gibson
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D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D017123:Bacteriophage T7; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D049308:Dioxygenases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010105:Oxygenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase