title: Kinetics and mechanism of oxygen-independent hydrocarbon hydroxylation by ethylbenzene dehydrogenase.
abstract: Ethylbenzene dehydrogenase (EBDH) from the denitrifying bacterium Azoarcus sp. strain EbN1 (to be renamed Aromatoleum aromaticum) catalyzes the oxygen-independent, stereospecific hydroxylation of ethylbenzene to (S)-1-phenylethanol, the first known example of direct anaerobic oxidation of a nonactivated hydrocarbon. The enzyme is a trimeric molybdenum/iron-sulfur/heme protein of 155 kDa that is quickly inactivated in air in its reduced state. Enzyme activity can be coupled to ferricenium tetrafluoroborate, providing a convenient way for kinetic measurements. EBDH exhibits activity with a wide 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 DeltaG298 for 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 theoretical considerations. Finally, the enzyme reaction has been established in an electrochemical reactor, allowing sustained enzymatic reaction and potential technical applications of the enzyme.
authors.affiliation: Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Szaleniec
authors.forename: Corina
authors.initials: C
authors.lastname: Hagel
authors.forename: Maurycy
authors.initials: M
authors.lastname: Menke
authors.forename: Pawel
authors.initials: P
authors.lastname: Nowak
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Witko
authors.forename: Johann
authors.initials: J
authors.lastname: Heider
mesh_terms: D020627:Azoarcus / Q000201:enzymology; D002384:Catalysis; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D007700:Kinetics; D008956:Models, Chemical; D008962:Models, Theoretical; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D004791:Enzyme Inhibitors; D010088:Oxidoreductases; C121602:ethylbenzene dehydrogenase; D010100:Oxygen