Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from Cycloclasticus sp. Strain A5

Abstract

ABSTRACT Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1 , phnA2 , phnA3 , and phnA4 genes, which encode the α and β subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the α and β subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.