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

TLDR: Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.

Abstract: Accidental spills and leaking underground storage tanks, as well as natural petroleum seeps, have released aromatic hydrocarbons into natural environments and led to abundant contamination of water resources (32). Aromatic hydrocarbons comprise one of the least reactive classes of organic molecules. Their relative inertness can be attributed to the absence of a functional group (e.g., hydroxyl, carbonyl, or carboxyl) and the resonance energy stabilization of the aromatic ring. These properties hamper rapid biodegradation of alkylbenzenes in many environments. However, some prokaryotic microorganisms employ intriguing metabolic strategies to activate alkylbenzenes leading to their oxidation in catabolic pathways. Under aerobic conditions, the well-characterized mono- and dioxygenases catalyze the activation of hydrocarbons by introducing a hydroxyl group via oxidative hydroxylation with molecular oxygen as cosubstrate (16). The absence of molecular oxygen in anoxic environments precludes this activation mode, and novel, alternative mechanisms are expected to operate under such conditions. In recent years, two mechanisms for initiating anaerobic metabolism of alkylbenzenes have emerged: methyl-substituted benzenes, such as toluene or m-xylene, are activated by enzymatic addition to fumarate to form benzylsuccinate or its methyl homolog (5, 6, 9, 25, 28, 34), whereas ethylbenzene (and possibly other alkylbenzenes with carbon chain length of ≥2) is oxidatively activated by an anaerobic dehydrogenation of the benzylic carbon to form 1-phenylethanol (3, 29). Ethylbenzene dehydrogenase is a novel enzyme in that it is the first enzyme shown to catalyze the hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen (Fig. ​(Fig.1).1). Stable isotope labeling studies showed that the hydroxyl group of 1-phenylethanol is derived from water (3). FIG. 1 Ethylbenzene dehydrogenase reaction as the first step in anaerobic ethylbenzene oxidation. In vitro studies with Azoarcus sp. strain EB1 demonstrated that ethylbenzene dehydrogenase activity is membrane associated and couples the oxidation of ethylbenzene to the reduction of p-benzoquinone (22). This dehydrogenation reaction is highly stereoselective and solely forms (S)-(−)-1-phenylethanol. The enzyme activity is expressed when Azoarcus sp. strain EB1 is grown anaerobically on ethylbenzene, as well as on 1-phenylethanol or acetophenone, as the sole carbon and electron source, but it is not expressed when cells are grown with benzoate. 1-Phenylethanol, acetophenone, and benzoate are intermediates in anaerobic ethylbenzene oxidation (3, 29). We report here for the first time on the purification and initial characterization of the novel ethylbenzene dehydrogenase, the cofactor content, and the nucleotide sequence and structure of the genes involved. Based on these findings, as well as on substrate transformation studies, we discuss possible reaction mechanisms for anaerobic ethylbenzene oxidation.