Benzylic and Aryl Hydroxylations of m-Xylene by the o-Xylene Dioxygenase from Rhodococcus sp. Strain DK17

Abstract

The o-xylene dioxygenase (XDO) from Rhodococcus sp. strain DK17 possesses the ability to perform distinct regioselective hydroxylations with the size and position of the substituent group determining the number and position of the dihydroxylation on the aromatic ring. Escherichia coli cells harboring a recombinant plasmid with the DK17 XDO genes were cotransformed with the pKJE7 chaperon plasmid for higher expression and stable maintenance of XDO, and then used for bioconversion of m&#8209

xylene . Gas chromatography-mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol as a major and a minor metabolite in a ratio of 8:2, respectively. A molecular modeling study suggests that the DK17 XDO holds xylene isomers at a kink region between α6 and α7 helices of the active site in a position, so that one side of the aromatic ring is blocked by helix α6. Although the substrate-binding pocket of XDO is modeled to be spacious when a xylene isomer binds as a substrate, m-xylene is unlikely to locate at the active site with a methyl group facing the kink region because such a configuration would not fit within the substrate-binding pocket. m-Xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3&#8209

methylbenzylalcohol could be formed as a product presumably due to the meta effect, which states that the meta methyl group of m-xylene would not allow binding and catalysis of aryl hydroxylation of XDO. Alternatively, m-xylene molecule can rotate counterclockwise from the previously mentioned position, which would allow the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol as a product. To our best knowledge, 2,4-dimethylphenol have not been seen before in the oxidation of m-xylene. The present work demonstrates that the DK17 o-xylene dioxygenase has an ability to oxidize m-xylene through both benzylic and aryl hydrxylations although the