Hydroxylation of bicyclic compounds by the o-xylene dioxygenase from Rhodococcus sp. strain DK17

Abstract

Rhodococcus sp. strain DK17 can metabolize various benzene derivatives including BTEX and bicyclic compounds through a common pathway initiated by o-xylene dioxygenase. The DK17 o-xylene dioxygenase enzyme possesses the unique ability to perform distinct regioselective hydroxylations that depend on the position and the size of the substituent groups on the aromatic ring. Bioconversion experiments were performed to further investigate the ability of the DK17 o-xylene dioxygenases to hydroxylate bulkier substrates including 1,2-dihydronaphthalene and biphenyl using the wild-type and mutant enzymes. Hydroxylation metabolites were identified and quantified by gas chromatography- mass spectrometry. The DK17 o-xylene dioxygenase hydroxylates 1,2-dihydronaphthalene at the 1,2 position of the alicyclic moiety to form cis-1,2-dihydroxy-1,2,3,4- tetrahydronaphthalene. Interestingly, one mutant enzyme (L266F) produced much more 2- hydroxybiphenyl (2.43 vs. 0.1 μg/L) and 3-hydroxybiphenyl (1.97 vs. 0.03 μg/L) than the wild-type. Site-directed mutagenesis combined with structural and functional analyses indicates that hydrophobic interactions and shielding effects against water are important factors in the hydroxylation of biphenyl by the o-xylene dioxygenase. In particular, the residue at position 266 plays a key role in coordinating the reaction.