The introduction of persistent intractable pollutants into the environment has created selective pressures by which microbes have developed the means to break down these xenobiotic compounds as sources of carbon and energy. Pseudomonas putida DLL-E4 is one such bacterium that has evolved the ability to break down para-nitrophenol. PnpC1C2, an enzyme in this catabolic pathway, oxidatively cleaves hydroquinone into 4-hydroxymuconic semialdehyde. PnpC1C2 belongs to the Type II hydroquinone dioxygenase family, a relatively uncharacterized class of mononuclear nonheme iron(II)-containing enzymes. Steady-state kinetics studies using UV/Vis spectroscopy were performed to characterize the enzyme specificity towards various substituted hydroquinones. In addition to its native substrate, PnpC1C2 showed a preference for monosubstituted hydroquinones. A variety of para-substituted phenols were explored as possible inhibitors of PnpC1C2. NMR studies were performed to determine the regiospecificity of ring cleavage with monosubstituted hydroquinones. These results showed notable differences for both the substrate specificity and ring-cleavage regiospecificity of PnpC1C2 compared to that of PcpA, a member of the Type I hydroquinone dioxygenase family, which is structurally related to well-studied catechol extradiol dioxygenase enzymes.

Characterization of PnpC1C2, a “type II” hydroquinone ring-cleaving dioxygenase

2019 Spring National Meeting

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