Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63.
Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min(-1) mg(-1)) and convert dibenzofuran to 2,2',3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.
Takagi
Terufumi
T
Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Japan.
Habe
Hiroshi
H
Yoshida
Takako
T
Yamane
Hisakazu
H
Omori
Toshio
T
Nojiri
Hideaki
H
D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases / Q000737:chemistry*; D004579:Electron Transport; D004926:Escherichia coli / Q000378:metabolism; D005288:Ferredoxins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D005288:Ferredoxins; D011994:Recombinant Proteins; D049308:Dioxygenases