Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the Gram-positive dibenzofuran-degrader Terrabacter sp strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade d ibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a pol ymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homolo gy to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activit y was confirmed in Escherichia coli cells containing the cloned dbfA1 and d bfA2 genes with the complementation of nonspecific ferredoxin and ferredoxi n reductase component of E. coli. Under this condition, these cells exhibit ed angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenox athiin. Phylogenetic analysis revealed that DbfA1 formed a branch with rece ntly reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxy genase from gram-positive bacteria but did not cluster with that of other a ngular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengau d, J., Happe, B., and Timmis, K. N. J. Bacteriol 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., N ojiri, H., Yamane, H., and Omori, T. J. Bacteriol 179, 4850-4858, 1997]. (C ) 2001 Academic Press.