Molecular characterization of Fdx1, a putidaredoxin-type [2Fe-2S] ferredoxin able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1

Abstract

Bacterium Sphingomonas sp. strain RW1 is, under aerobic conditions, able to degrade dibenzofuran and dibenzo-p-dioxin, The first step of the pathway is performed by a ring-dihydroxylating enzyme. Bunz and Cook have reported the purification and characterization of this dioxin dioxygenase and a ferredoxin able to transfer electrons to the dioxygenase [Bunz, P. V. and Cook, A. M. (1993) J. Bacteriol. 175, 6467-6475]. The gene encoding this [2Fe-2S] ferredoxin was identified by screening a genomic library constructed in pLAFR3 with a probe generated by a nested-PCR amplification. Primers for the amplification were designed based on the N-terminus sequence of the purified ferredoxin and on sequence comparisons with related proteins. Several cosmids were obtained and the ferredoxin gene, fdx1, was subcloned from one of them. The nucleotide sequence of a 4.6-kb DNA fragment encompassing the ferredoxin gene was determined. While in the case of all known multi-component dioxygenases, genes encoding the a and β subunits are found to be contiguous with the gene of the specific electron carrier, the fdx1 gene in Sphingomonas sp. RW1 does not appear to be directly linked with the dioxin dioxygenase genes. Rather, it is clustered with genes apparently encoding two atypical decarboxylases/isomerases and a glutathione S-transferase. The ferredoxin gene was hyperexpressed and the recombinant ferredoxin was purified. Spectroscopic characterization of Fdx1 demonstrated the presence of a putidaredoxin-type [2Fe-2S] cluster in this protein. Its redox potential was determined to be -245 (± 5) mV versus the normal hydrogen electrode at 25°C, pH 8.0. Therefore, the protein is closely related to [2Fe-2S] ferredoxins known to be electron donors to monooxygenases involved in hydroxylation of aromatic compounds. Thus, this report provides clear evidence that a putidaredoxin-type [2Fe-2S] ferredoxin, namely Fdx1, is able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1.