title: In vitro studies on the initial reactions of anaerobic ethylbenzene mineralization.
abstract: Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) [mg of protein](-1) and an apparent K(m) for ethylbenzene of approximately 60 microM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.
authors.affiliation: Environmental Engineering and Science, Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305-4020, USA.
authors.forename: H A
authors.initials: HA
authors.lastname: Johnson
authors.forename: A M
authors.initials: AM
authors.lastname: Spormann
mesh_terms: D000693:Anaerobiosis; D001555:Benzene Derivatives / Q000378:metabolism*; D001592:Benzyl Alcohols / Q000378:metabolism; D001673:Biodegradation, Environmental; D004156:Catechol Oxidase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006089:Gram-Negative Anaerobic Bacteria / Q000378:metabolism*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010105:Oxygenases; D013379:Substrate Specificity
chemical_list: D001555:Benzene Derivatives; D001592:Benzyl Alcohols; C002017:methylphenyl carbinol; D010088:Oxidoreductases; D008291:Malate Dehydrogenase; D004156:Catechol Oxidase; D010105:Oxygenases; C121603:1-phenylethanol dehydrogenase; C121602:ethylbenzene dehydrogenase; C004912:ethylbenzene