Dihydroxylation of four- and five-ring aromatic hydrocarbons by the naphthalene dioxygenase from Sphingomonas CHY-1.

Abstract

The naphthalene dioxygenase from Sphingomonas CHY-1 exhibits extremely broad substrate specificity toward polycyclic aromatic hydrocarbons (PAHs). In a previous study, the catalytic rates of oxidation of nine PAHs were determined using the purified dioxygenase, but the oxidation products formed from four- to five-ring hydrocarbons were incompletely characterized. Here, we reexamined PAH oxygenation reactions using Escherichia coli recombinant cells overproducing strain CHY-1 dioxygenase. Hydroxylated products generated by the dioxygenase were purified and characterized by means of GC-MS, UV absorbance as well as 1H- and 13C-NMR spectroscopy. Fluoranthene was converted to three dihydrodiols, the most abundant of which was identified as cis-7,8-dihydroxy-7,8-dihydrofluoranthene. This diol turned out to be highly unstable, converting to 8-hydroxyfluoranthene by spontaneous dehydration. The dioxygenase also catalyzed dihydroxylations on the C2-C3 and presumably the C1-C2 positions, although at much lower rates. Benz[a]anthracene was converted into three dihydrodiols, hydroxylated in positions C1-C2, C8-C9, and C10-C11, and one bis-cis-dihydrodiol. The latter compound was identified as cis,cis-1,2,10,11-tetrahydroxy-1,2,10,11-tetrahydrobenz[a]anthracene, which resulted from the subsequent dioxygenation of the 1,2- or 10,11-dihydrodiols. Chrysene dioxygenation yielded a single diol identified as cis-3,4-dihydroxy-3,4-dihydrochrysene, which underwent further oxidation to give cis,cis-3,4,9,10 chrysene tetraol. Pyrene was a poor substrate for the CHY-1 dioxygenase and gave a single dihydrodiol hydroxylated on C4 and C5, whereas benzo[a}pyrene was converted to two dihydrodiols, one of which was identified as cis-9,10-dihydrodiol. The selectivity of the dioxygenase is discussed in the light of the known 3D structure of its catalytic component and compared to that of the few enzymes able to attack four- and five-ring PAHs.