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Journal of Bacteriology, August 2001, p. 4536-4542, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4536-4542.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme

Hope A. Johnson,1 Dale A. Pelletier,1 and Alfred M. Spormann1,2,*

Environmental Engineering and Science, Department of Civil and Environmental Engineering,1 and Department of Biological Sciences,2 Stanford University, Stanford, California 94305-4020

Received 2 February 2001/Accepted 27 April 2001

The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alpha beta gamma ) with subunit masses of 100 kDa (alpha ), 35 kDa (beta ), and 25 kDa (gamma ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the alpha  subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the beta  subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the gamma  subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.

* Corresponding author. Mailing address: Environmental Engineering and Science, Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305-4020. Phone: (650) 723-3668. Fax: (650) 725-3164. E-mail: spormann{at}stanford.edu.

Journal of Bacteriology, August 2001, p. 4536-4542, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4536-4542.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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