Sequencing and functional analysis of a multi-component dioxygenase from PAH-degrading Sphingomonas paucimobilis EPA505

Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic organic compounds consisting of two or more fused benzene rings. PAHs derive from many different sources including petroleum refining, wood treatment, and coal coking industries. Because of their structural stability and water insolubility, PAHs are extremely resistant to degradation. These compounds are also believed to have mutagenic, carcinogenic, and teratogenic effects. Therefore, there are currently 16 PAH compounds on the EPA's list of priority pollutants.;Many species of bacteria have the ability to breakdown these persistent pollutants. However, bioremediation strategies using these organisms have many unresolved issues. While laboratory experiments can easily demonstrate the ability of these organisms to breakdown pollutants, environmental factors may reduce degradation abilities in situ.;Within the prokaryotes, members of the genus Sphingomonas have demonstrated a greater ability to breakdown PAHs. Spingomonas paucimobilis EPA505, for example, was shown to degrade a wide range of PAHs including the high molecular weight PAH fluoranthene, which it could also use as a sole carbon source. Because of its potential as a bioremediation tool, it is important to study the molecular basis of PAH catabolism in EPA505.;A genomic library of EPA505 was constructed and probed for genes involved in PAH degradation. Complete gene sequences were obtained for four subunits which are involved in the first step of the PAH catabolism. This step is catalyzed by a dioxygenase enzyme and yields a dihydrodiol intermediate. Two of the gene sequences encode an alpha and beta subunit of the dioxygenase. The third gene encodes a ferredoxin subunit and the fourth gene codes for a ferredoxin reductase subunit.;The four genes were cloned for expression. Expression host cells were induced to test the activity of the four recombinant proteins on various PAHs. When cells expressing all four subunits were incubated with naphthalene and phenanthrene, the corresponding dihydrodiol product was detected using GC-MS. No dihydrodiol product was detected when fluoranthene was tested. In addition, no dihydrodiol products were detected for any substrate when cells lacking the two ferredoxin subunits were tested.;This study identified and showed functional analysis of one enzyme, a PAH degrading dioxygenase in the PAH catabolic pathway of Sphingomonas paucimobilis EPA505. There is still much to learn in order to fully appreciate and take advantage of this organism as an efficient tool for bioremediation.