title: The reductase RedA2 of the multi-component dioxin dioxygenase system of Sphingomonas sp. RW1 is related to class-I cytochrome P450-type reductases.
abstract: The first step in the oxidation of the diaryl ethers dibenzo-p-dioxin and dibenzofuran by the bacterium Sphingomonas sp. RW1 is carried out by an atypical multi-component ring hydroxylating dioxygenase. This heteromeric enzyme requires the participation of a flavoprotein, reductase A2, and an iron-sulfur protein, Fdx1, to mediate the transfer of electrons from NADH to the dioxygenase for oxygen activation [Bünz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467-6475]. From the type of ferredoxin (Fd) and flavoprotein, this complex is presumed to belong to the class-IIA dioxygenase system which has not been genetically analysed so far. The gene encoding the flavoprotein was identified by screening a genomic library constructed in pLAFR3 with a probe generated by PCR amplification. The nucleotide sequence of a 2.0-kb DNA fragment encompassing the reductase gene, redA2, was determined. The specified protein shares 37-40% identity with class-I cytochrome P450 reductases and 27-35% identity with reductases acting with class-IIB dioxygenases. An FAD-binding amino acid consensus sequence, as well as an NADH-binding site were detected by analogy beginning at residues 10 and 153, respectively. The redA2 gene is not linked to the dioxin dioxygenase cistrons. The rare start codon, GTG, of the reductase was changed to ATG and the modified gene hyperexpressed in Escherichia coli using the strong T7 polymerase promoter. The recombinant reductase was purified to homogeneity with an approximate yield of 3.3 mg/g wet mass cells. Flavin analysis confirmed the presence of 1 FAD/mol protein. The Km values for NADH and Fdx1 are 22 (+/- 3) microM and 3.7 (+/- 0.4) microM, respectively.
authors.affiliation: Division of Microbiology, GBF - National Research Centre for Biotechnology, Braunschweig, Germany. Jar@gbf.de
authors.forename: J
authors.initials: J
authors.lastname: Armengaud
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000235:genetics*; D010105:Oxygenases / Q000235:genetics*; D010802:Phylogeny; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C113047:RedA2 reductase; D010105:Oxygenases; C108163:dioxin dioxygenase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase