Ethylbenzene dehydrogenase (EBDH) from the denitrifying bacterium
Azoarcus sp. strain EbN1(to be renamed
Aromatoleum aromaticum) catalyzes the oxygen-independent, stereospecific hydroxylationof ethylbenzene to (
S)-1-phenylethanol, the first known example of direct anaerobic oxidation of anonactivated hydrocarbon. The enzyme is a trimeric molybdenum/iron-sulfur/heme protein of 155 kDathat is quickly inactivated in air in its reduced state. Enzyme activity can be coupled to ferriceniumtetrafluoroborate, providing a convenient way for kinetic measurements. EBDH exhibits activity with awide range of ethylbenzene analogues, which were analyzed for their kinetic parameters, stoichiometry,and formed products. The reactivity was correlated to the chemical structures by a quantitative structure-activity relationship (QSAR) model. On the basis of these results, quantum chemical calculations of
G298for formation of carbocations of the respective substrates were performed and used in reactivity analysis.A putative reaction mechanism is proposed on the basis of the experimental results and theoreticalconsiderations. Finally, the enzyme reaction has been established in an electrochemical reactor, allowingsustained enzymatic reaction and potential technical applications of the enzyme.