THE REDUCTASE REDA2 OF THE MULTICOMPONENT DIOXIN DIOXYGENASE SYSTEM OF SPHINGOMONAS SP. RW1 IS RELATED TO CLASS-I CYTOCHROME P-450-TYPE REDUCTASES

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 he teromeric enzyme requires the participation of a flavoprotein, reducta se A2, and an iron-sulfur protein, Fdx1, to mediate the transfer of el ectrons from NADH to the dioxygenase for oxygen activation [Bunz, 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 ana lysed so far. The gene encoding the flavoprotein was identified by scr eening 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 P-450 reductas es and 27-35% identity with reductases acting with class-IIB dioxygena ses. 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 nias purifie d 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 K-m values for NADH and Fdx1 are 22 (+/-3) mu M and 3.7 (+/-0.4) mu M, res pectively.