Preparation of dihydroxy polycyclic aromatic hydrocarbons and activities of two dioxygenases in the phenanthrene degradative pathway

Dihydroxy phenanthrene, fluoranthene, and pyrene derivatives are intermediates in the bacterial catabolism of the corresponding parent polycyclic aromatic hydrocarbon (PAH). Ring-opening of the dihydroxy species followed by a series of enzyme-catalyzed reactions generates metabolites that funnel into the Krebs Cycle with the eventual production of carbon dioxide and water. One complication in delineating these pathways and harnessing them for useful purposes is that the initial enzymatic processing produces multiple dihydroxy PAHs with multiple ring opening possibilities and products. As part of a systematic effort to address this issue, eight dihydroxy species were synthesized and characterized as the dimethoxy or diacetate derivatives. Several dihydroxy compounds were examined with two dioxygenases in the phenanthrene degradative pathway in Mycobacterium vanbaalenii PYR-1. One, 3,4-dihydroxyphenanthrene, was processed by PhdF with a k_cat/K_m of 6.0 × 10⁶ M^-1s^-1, a value that is consistent with the annotated function of PhdF in the pathway. PhdI processed 1-hydroxy-2-naphthoate with a k_cat/K_m of 3.1 × 10⁵ M^-1s^-1, which is also consistent with the proposed role in the pathway. The observations provide the first biochemical evidence for these two reactions in M. vanbaalenii PYR-1 and, to the best of our knowledge, the first biochemical evidence for the reaction of PhdF with 3,4-dihydroxyphenanthrene. Although PhdF is upregulated in the presence of pyrene, it did not process two dihydroxypyrenes. Methodology was developed for product analysis of the extradiol dioxygenases.