Identification of three novel salicylate 1-hydroxylases involved in the phenanthrene degradation of Sphingobium sp. strain P2.

Five sets of large and small subunits of terminal oxygenase (ahdA1[a-e] and ahdA2[a-e]) and a single gene set encoding ferredoxin (ahdA3) and ferredoxin reductase (ahdA4) were found to be scattered through 15.8- and 14-kb DNA fragments of phenanthrene-degrading Sphingobium sp. strain P2. RT-PCR analysis indicated the inducible and specific expression of ahdA3, ahdA4, and three sets of genes for terminal oxygenase (ahdA1[c-e] and ahdA2[c-e]) in this strain grown on phenanthrene. The biotransformation experiments with resting cells of Escherichia coli JM109 harboring recombinant ahd genes revealed that AhdA2cA1c, AhdA1dA2d, and AhdA1eA2e can all function as a salicylate 1-hydroxylase which converts salicylate, a metabolic intermediate of phenanthrene, to catechol in cooperation with the electron transport proteins AhdA3A4. The first two oxygenases exhibited a broad range of substrate specificities such that they also catalyzed the hydroxylation of methyl- and chloro-substituted salicylates to produce their corresponding substituted catechols.