Isolation and characterization of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme

Abstract

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 (alphabetagamma) 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.

FIG. 1

Ethylbenzene dehydrogenase reaction as the first step in anaerobic ethylbenzene oxidation.

FIG. 2

SDS-PAGE of the purified ethylbenzene dehydrogenase from Azoarcus sp. strain EB1. Lane 1, molecular mass standards in kilodaltons; lane 2, 0.5 μg of ethylbenzene dehydrogenase protein recovered from a cation-exchange column.

FIG. 3

Fluorescence spectra of purified ethylbenzene dehydrogenase (1 mg ml⁻¹) prepared in the presence (A_ex and A_em, solid lines) or absence (B_ex and B_em, dashed lines) of iodine. The emission spectra (A_em and B_em) were recorded at 375 nm. The excitation spectra (A_ex and B_ex) were recorded at 470 nm.

FIG. 4

Organization of the genes encoding anaerobic ethylbenzene dehydrogenase from Azoarcus sp. strain EB1. Arrows indicate the direction of transcription.